Microbial derived isoprene and methods for making the same

ABSTRACT

Provided herein is a gaseous isoprene composition comprising isoprene, carbon dioxide and water, wherein the isoprene is in an amount between about 0.1% and about 15% by volume; wherein the carbon dioxide is in an amount between about 0.04% and about 35% by volume; wherein the water is in an amount greater than about 70% of its saturation amount. Also provided herein is a liquid isoprene composition comprising isoprene in an amount of at least 65% by weight and carbon dioxide in an amount between about 0.01% and about 1% by weight.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation application of the U.S. Non-Provisionalapplication Ser. No. 14/956,402, filed on Dec. 2, 2015, which is adivisional application of the U.S. Non-Provisional application Ser. No.13/887,381, filed on May 6, 2013, now U.S. Pat. No. 9,233,894, which isa divisional application of the U.S. Non-Provisional application Ser.No. 13/629,623, filed on Sep. 28, 2012, now U.S. Pat. No. 8,492,605,which is a divisional application of the U.S. Non-Provisionalapplication Ser. No. 12/659,216, filed on Mar. 1, 2010, now U.S. Pat.No. 8,324,442, which claims the benefit of priority from U.S.Provisional Application No. 61/202,474, filed on Mar. 3, 2009, all ofwhich in their entirety are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Isoprene is a five carbon hydrocarbon (2-methyl-1,3-butadiene), that isan industrial chemical used in a range of industrial application such astires, footwear, sporting goods, latex, tapes, labels, and medicaldisposables. Isoprene is also a natural compound produced in biologicalsystems. While isoprene is made naturally in various organisms rangingfrom microbes to animals, most naturally occurring isoprene hastraditionally been extracted from rubber plants. However, extractionyields are low and these quantities are far less than are required formany commercial applications. As a result, isoprene is primarilyproduced synthetically from petroleum sources, most often from ethyleneusing a steam cracking process.

Due to the growing concern for climate change and thus a need to makeproducts we need more sustainably, there is an urgent need for bio- orrenewable isoprene that will help meet global isoprene demands but thatcan be produced in a more environmentally friendly way. The currentinvention addresses this need.

SUMMARY OF THE INVENTION

The present invention provides microbial derived isoprene compositionsand methods for making and purifying the same.

In one aspect of the invention, a gaseous isoprene composition isprovided comprising: isoprene, carbon dioxide and water, wherein theisoprene is in an amount between about 0.1% and about 15% by volume;wherein the carbon dioxide is in an amount between about 0.04% and about35% by volume; wherein the water is in an amount greater than about 70%of its saturation amount; and wherein the gaseous isoprene compositioncomprises 1 part per million or less than 1 part per million of any oneof the following impurities: C₂-C₅ alkynes, cyclopentadiene, piperylene,and 1,4-pentadiene.

In some embodiments, the gaseous composition comprises less than about3% by weight of water. In certain embodiments, the gaseous compositionfurther comprises oxygen in an amount between about 1% and about 20% byvolume. In some embodiments, the gaseous composition further comprisesnitrogen in an amount greater than about 50% by volume. In certainembodiments, the gaseous composition further comprises argon in anamount less than about 0.9% by volume or greater than about 1.0% byvolume. In certain embodiments, the gaseous composition furthercomprises ethanol in an amount less than about 0.5% by volume or morethan about 1% by volume. In certain embodiments, the gaseous compositionfurther comprises 1 part per million or less of cyclopentadiene,piperylene, 1,4-pentadiene, or a combination thereof.

In another aspect a liquid isoprene composition is provided comprising:isoprene in an amount of at least 65% by weight and carbon dioxide in anamount between about 0.01% and about 1% by weight, wherein the isoprenecomposition comprises 1 part per million or less than 1 part per millionof any one of the following impurities: C₂-C₅ alkynes, cyclopentadiene,piperylene, and 1,4-pentadiene.

In some embodiments, the liquid isoprene composition disclosed hereincomprises at least about 80% or at least about 95% isoprene by weight.In some embodiments, the liquid isoprene composition comprises less thanabout 1% by weight of water. In certain embodiments, the liquid isoprenecomposition further comprises nitrogen in an amount between about 0.001%and about 1% by weight. In some embodiments, the liquid isoprenecomposition further comprises ethanol in an amount greater than about0.01% by weight. In certain embodiments, the carbon dioxide is in anamount between about 0.05% and about 1% by weight, or between about 0.1%and about 1% by weight, or between about 0.2% and about 0.7% by weight.

In another aspect, a method for making and purifying isoprene isprovided. The method comprises:

-   -   a. obtaining a first gaseous composition comprising isoprene and        water wherein the gaseous composition comprises 1 part per        million or less of C₂-C₅ alkynes;    -   b. flowing the first gaseous composition through a first chiller        wherein the first chiller has a temperature of between about        10° C. and about −15° C. thereby resulting in a second gaseous        composition and wherein the second gaseous composition comprises        less water than the first gaseous composition;    -   c. flowing the second gaseous composition through a second        chiller wherein the second chiller has a temperature below −35°        C.; and    -   d. collecting the resulting liquid isoprene composition.

In another aspect, another method is provided. The method comprises:

-   -   a. culturing a plurality of host cells capable of making        isoprene;    -   b. forming a first gaseous composition comprising isoprene and        water wherein the water is present in an amount greater than        about 70% of its saturation amount;    -   c. subjecting the first gaseous composition to a first cooling        step whereby substantially all of the water is removed from the        first gaseous composition resulting a second gaseous        composition; and    -   d. subjecting the second gaseous composition to a second cooling        step whereby a liquid isoprene composition is collected.

In another aspect, another method is provided. The method comprises:

-   -   a. culturing a plurality of host cells capable of making        isoprene;    -   b. forming a first gaseous composition comprising isoprene and        water wherein the water is present in an amount greater than        about 70% of its saturation amount;    -   c. subjecting the first gaseous composition to a first cooling        step whereby substantially all of the water is removed from the        first gaseous composition resulting a second gaseous        composition;    -   d. subjecting the second gaseous composition to a second cooling        step whereby a liquid isoprene composition is collected; and    -   e. optionally, contacting either the first gaseous composition,        the second gaseous composition and/or the liquid isoprene        composition with a membrane containing modified zeolites or        molecular sieve to provide a purified isoprene composition.

In another aspect, another method is provided. The method comprises:

-   -   a. contacting a plurality of host cells capable of making        isoprene in an aqueous medium wherein the aqueous medium is in        contact with an immiscible organic liquid and the aqueous        medium, the host cells, and the immiscible organic liquid is in        a closed vessel; and    -   b. culturing the host cells in the aqueous medium whereby the        host cells make isoprene and the isoprene is captured in the        immiscible organic liquid.

In another aspect, another method is provided. The method comprises:

-   -   a. obtaining a first gaseous composition comprising:        -   i. isoprene in an amount between about 0.1% and about 15% by            volume;        -   ii. carbon dioxide in an amount between about 0.04% and            about 35% by volume;        -   iii. oxygen in an amount between about 1% and about 20% by            volume;        -   iv. nitrogen in an amount greater than about 50% by volume;        -   v. argon in an amount less than about 0.9% by volume;        -   vi. water in an amount greater than about 70% of its            saturation amount;        -   vii. 1 part per million or less of C2-C5 alkyne,            cyclopentadiene, piperylene, and 1,4-pentadiene; and        -   viii. ethanol;    -   b. flowing the first gaseous composition through a first chiller        and an operably connected flash drum, wherein the first chiller        has a temperature of between about 10° C. and about −15° C.        thereby resulting in a second gaseous composition and wherein        the second gaseous composition comprises less water than the        first gaseous composition;    -   c. flowing the second gaseous composition through a second        chiller and an operably connected flash drum, wherein the second        chiller has a temperature between about 35° C. and about −85°        C.; and    -   d. collecting the resulting liquid isoprene composition.

An isoprene production system comprising:

-   -   a. a bioreactor capable of culturing a plurality of host cells;    -   b. a first chiller and flash drum operably connected to the        overhead stream of the bioreactor, the first chiller capable of        operating in a temperature range of between 10° C. and −15° C.;        and    -   c. a second chiller and flash drum operably connected to the        overhead stream exiting from the first chiller and flash drum,        the second chiller capable of operating in a temperature below        −35° C.

In yet another aspect, an isoprene production system is provided. Thesystem comprises:

-   -   a. a closed vessel;    -   b. an aqueous medium, within the vessel, forming a first phase;    -   c. a plurality of host cells, within the aqueous medium, capable        of making isoprene; and,    -   d. a liquid organic second phase, capable of capturing the        isoprene made by the host cells, in contact with the first        phase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of an exemplary separation system.

FIG. 2 is another schematic representation of another exemplaryseparation system.

FIG. 3 is a schematic representation of the mevalonate (“MEV”) pathwayfor the production of isopentenyl diphosphate (“IPP”).

FIG. 4 is a schematic representation of the DXP pathway for theproduction of IPP and dimethylallyl pyrophosphate (“DMAPP”). Dxs is1-deoxy-D-xylulose-5-phosphate synthase; Dxr is1-deoxy-D-xylulose-5-phosphate reductoisomerase (also known as IspC);IspD is 4-diphosphocytidyl-2C-methyl-D-erythritol synthase; IspE is4-diphosphocytidyl-2C-methyl-D-erythritol synthase; IspF is2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG is1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG); and ispHis isopentenyl/dimethylallyl diphosphate synthase.

FIG. 5 shows the recovery of isoprene from an immiscible organic liquid(isopropyl myristate) from a closed fermentation system.

FIG. 6 shows a map of plasmid pAM1547.

FIG. 7A-FIG. 7G show maps of the inserts of vectors pAM489, pAM491,pAM493, pAM495, pAM497, and pAM584, and of the integration cassettenatA-P_(CTR3) ^(−1 to −734.)

FIG. 8 shows a map of the pMULE Entry vector.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Reference is made here to anumber of terms that shall be defined to have the following meanings:

“Bio-organic compound” refers to an organic compound having at leastfive carbon atoms that can be made by a host cell by taking acarbohydrate carbon source and converting the carbohydrate carbon sourceinto the desired product.

“Deoxyxylulose 5-phosphate pathway” or “DXP pathway” is used herein torefer to the pathway that converts glyceraldehyde-3-phosphate andpyruvate to IPP and DMAPP. The DXP pathway is illustrated schematicallyin FIG. 4.

“Endogenous” refers to a substance or process that can occur naturally,e.g., in a non-recombinant host cell.

“Heterologous nucleic acid” as used herein refers to a nucleic acidwherein at least one of the following is true: (a) the nucleic acid isforeign (“exogenous”) to (that is, not naturally found in) a given hostcell; (b) the nucleic acid comprises a nucleotide sequence that isnaturally found in (that is, is “endogenous to”) a given host cell, butthe nucleotide sequence is produced in an unnatural (for example,greater than expected or greater than naturally found) amount in thecell; (c) the nucleic acid comprises a nucleotide sequence that differsin sequence from an endogenous nucleotide sequence, but the nucleotidesequence encodes the same protein (having the same or substantially thesame amino acid sequence) and is produced in an unnatural (for example,greater than expected or greater than naturally found) amount in thecell; or (d) the nucleic acid comprises two or more nucleotide sequencesthat are not found in the same relationship to each other in nature (forexample, the nucleic acid is recombinant).

“Host cell” and “microorganism” are used interchangeably herein to referto any archae, bacterial, or eukaryotic living cell into which aheterologous nucleic acid can be or has been inserted. The term alsorelates to the progeny of the original cell, which may not necessarilybe completely identical in morphology or in genoic or total DNAcomplement to the original parent, due to natural, accidental, ordeliberate mutation.

“Isoprenoid” and “isoprenoid compound” are used interchangeably hereinand refer to a compound derivable from isopentenyl diphosphate.

“Isolate” and “isolating” when referred to a bio-organic compound is theenrichment of the amount of the bio-organic compound in a composition.Consequently, the amount of the bio-organic compound in a compositionafter the bio-organic compound has been isolated or subject to anisolating step is greater than the amount present in the compositionprior to such step.

“Mevalonate pathway” or “MEV pathway” is used herein to refer to thebiosynthetic pathway that converts acetyl-CoA to IPP. The MEV pathway isillustrated schematically in FIG. 3.

“Naturally occurring” as applied to a nucleic acid, an enzyme, a cell,or an organism, refers to a nucleic acid, enzyme, cell, or organism thatis found in nature. For example, a polypeptide or polynucleotidesequence that is present in an organism that can be isolated from asource in nature and that has not been intentionally modified by a humanin the laboratory is naturally occurring.

“Optional” or “optionally” means that the subsequently described featureor structure may or may not be present, or that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where a particular feature or structureis present and instances where the feature or structure is absent, orinstances where the event or circumstance occurs and instances where theevent or circumstance does not occur.

“Pyrophosphate” is used interchangeably herein with “diphosphate”.

As used herein, a composition that is a “substantially pure” compound issubstantially free of one or more other compounds, i.e., the compositioncontains greater than 80 vol. %, greater than 90 vol. %, greater than 95vol. %, greater than 96 vol. %, greater than 97 vol. %, greater than 98vol. %, greater than 99 vol. %, greater than 99.5 vol. %, greater than99.6 vol. %, greater than 99.7 vol. %, greater than 99.8 vol. %, greaterthan 99.9 vol. % of the compound; or less than 20 vol. %, less than 10vol. %, less than 5 vol. %, less than 4 vol. %, less than 3 vol. %, lessthan 2 vol. %, less than 1 vol. %, less than 0.5 vol. %, less than 0.1vol. %, or less than 0.01 vol. % of the one ore more other compounds,based on the total volume of the composition.

In the following description, all numbers disclosed herein areapproximate values, regardless whether the word “about” or “approximate”is used in connection therewith. They may vary by 1 percent, 2 percent,5 percent, or, sometimes, 10 to 20 percent. Whenever a numerical rangewith a lower limit, RL and an upper limit, RU, is disclosed, any numberfalling within the range is specifically disclosed. In particular, thefollowing numbers within the range are specifically disclosed:R=RL+k*(RU−RL), wherein k is a variable ranging from 1 percent to 100percent with a 1 percent increment, i.e., k is 1 percent, 2 percent, 3percent, 4 percent, 5 percent, . . . , 50 percent, 51 percent, 52percent, . . . , 95 percent, 96 percent, 97 percent, 98 percent, 99percent, or 100 percent. Moreover, any numerical range defined by two Rnumbers as defined in the above is also specifically disclosed.

In addition to the definitions above, certain compounds described hereinhave one or more double bonds that can exist as either the Z or Eisomer. The invention in certain embodiments encompasses these compoundsas individual isomers in a substantially pure form as well as mixturesof various isomers, e.g., racemic mixtures of stereoisomer.

Current Sources of Isoprene

Isoprene currently is made naturally by rubber plants (typically Heveabrasiliensis) or is made synthetically from petroleum sources. When madenaturally, the sap-like extract (known as latex and is a polymerizedversion of isoprene) is collected from the rubber plants and is theprimary source of natural rubber. Because latex and natural rubber canbe of varying quality (irregular molecular distribution), the syntheticanalog of natural rubber or polyisoprene is often preferred due to itshigher uniformity.

Chemically synthesized isoprene is made primarily from petroleumsources. The most common method involves stream cracking a petroleumstream to make ethylene which in turn is subsequently converted intoisoprene. Other methods for making isoprene include isobutylenecarbonylation and isopentane dehydrogenation. The resulting isoprene isproduced and sold in different concentrations. Crude isoprene has apurity between 15% and 65%. Refined isoprene is defined as isoprenehaving a purity between 65% and 95%. High purity isoprene is defined asisoprene having a purity between 95% and 99.5%. Polymer grade isopreneis isoprene with a purity exceeding 99.5%.

As a consequence of how it is made, synthetic isoprene contains a numberof impurities including various acetylenes and dienes such ascyclopentadiene and piperylene. Although these catalysts are undesirableas they inhibit polymerization, it is not often economical to entirelyeliminate them and the purity of the isoprene is matched to the desiredend product. For example, the isoprene purity required to make butylrubber is substantially less that required to make SIS polymers (polymergrade).

Microbially Derived Gaseous Isoprene Compositions

The present invention provides microbial derived isoprene compositionsand methods for making and purifying the same. Microbial-derivedisoprene compositions differ from petroleum derived sources in that thecompositions include virtually none of the following impurities: C₂-C₅alkynes; cyclopentadiene, piperylene and 1,4-pentadiene.

In one aspect of the invention, a gaseous isoprene composition isprovided. The composition comprises isoprene and water wherein the wateris present in an amount that is at least about 70% of its saturationamount and the composition comprises 1 part per million or less of C₂-C₅alkynes. Illustrative examples of C₂-C₅ alkynes include acetylene,isopropylacetylene, 1-pentyne, 2-pentyne, and 2-butyne.

In another aspect of the invention, another gaseous isoprene compositionis provided. The composition comprises isoprene and water wherein thewater is present in an amount that is at least about 70% of itssaturation amount and the composition comprises 1 part per million orless of cyclopentadiene.

In another aspect of the invention, another gaseous isoprene compositionis provided. The composition comprises isoprene and water wherein thewater is present in an amount that is at least about 70% of itssaturation amount and the composition comprises 1 part per million orless of piperylene.

In another aspect of the invention, another gaseous isoprene compositionis provided. The composition comprises isoprene and water wherein thewater is present in an amount that is at least about 70% of itssaturation amount and the composition comprises 1 part per million orless of 1,4-pentadiene.

In another aspect of the invention, another gaseous isoprene compositionis provided. The composition comprises isoprene and water wherein thewater is present in an amount that is at least about 70% of itssaturation amount and the composition comprises 1 part per million orless of each of C₂-C₅ alkynes, cyclopentadiene, piperylene, and1,4-pentadiene.

In some embodiments, the gaseous isoprene composition comprises isoprenethat is present between about 0.1% and about 15% by volume. In otherembodiments, the isoprene is present between about 1 and 10% by volume.In still other embodiments, the isoprene is present between about 1 and5% by volume. In yet other embodiments, the isoprene is present betweenabout 5% and about 10% by volume. In further embodiments, the isopreneis present between in an amount greater than about 10% by volume.

In other embodiments, the gaseous isoprene composition comprises waterin an amount that is greater than about 70%, 75%, 80%, 85%, 90%, 95% and99% of its saturation amount. In still other embodiments, the gaseousisoprene composition comprises saturated water.

In other embodiments, the gaseous isoprene composition further comprisescarbon dioxide that is present in an amount that is greater than about0.04% by volume. In still other embodiments, the carbon dioxide ispresent in an amount that is greater than about 0.05%, 0.1%, 0.5%, 1.0%,and 5% by volume. In further embodiments, the carbon dioxide is presentin an amount that is greater than about 10%, about 20%, about 30% byvolume. In still further embodiments, the carbon dioxide is present inan amount that is between about 1% and about 35% by volume. In stillother embodiments, the carbon dioxide is present in an amount that isbetween about 10% and about 30% by volume.

In other embodiments, the gaseous isoprene composition further comprisesoxygen. In some embodiments, the oxygen is present in an amount that isless than about 20.9% by volume. In other embodiments, the oxygen ispresent in an amount that is between about 1% by volume and about 20% byvolume. In other embodiments, the oxygen is present in an amount that isbetween about 8% and about 15% by volume. In other embodiments, theoxygen is present in an amount that is less than about 20%, 15%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, and 2%. In still other embodiments, theoxygen is present in an amount that is less than about 1% by volume. Infurther embodiments, the oxygen is present between about 1% and about15% by volume. In still further embodiments, the oxygen is presentbetween about 5% and about 15% by volume.

In other embodiments, the gaseous isoprene composition further comprisesnitrogen. In some embodiments, the nitrogen is present in an amountbetween about 50% and about 75% by volume. In further embodiments, thenitrogen is present in an amount that is greater than about 70%. Inother embodiments, the nitrogen is present in an amount that is greaterthan about 75%, 76%, 77%, 78%, 79%, and 80%.

In other embodiments, the gaseous isoprene composition further comprisesargon. In some embodiments, the argon is present in an amount that isless than about 0.9% by volume. In other embodiments, the argon ispresent in an amount that is greater than about 1.0% by volume.

In other embodiments, the gaseous isoprene composition further comprisesethanol. In some embodiments, the ethanol is present in an amount thatis less than about 0.5% by volume. In other embodiments, the ethanol ispresent in an amount that is more than about 1% by volume.

In other embodiments, the microbial-derived gaseous isoprene compositionmay comprise: isoprene in an amount between about 0.1% and about 15% byvolume; water in an amount that is greater than about 70% of itssaturation amount; carbon dioxide in an amount that is between about0.04% and about 35% by volume; oxygen in an amount that is between about1% and about 20% by volume; nitrogen in an amount that is greater thanabout 50% by volume; argon in an amount that is less than about 0.9% byvolume; ethanol in an amount that is less than about 0.5% by volume; 1part per million or less of C₂-C₅ alkynes; 1 part per million or less ofcyclopentadiene; 1 part per million or less of piperylene; and 1 partper million or less of 1,4-pentadiene.

In other embodiments, the microbial-derived gaseous isoprene compositionmay comprise: isoprene in an amount between about 0.1% and about 15% byvolume; water in an amount that is greater than about 70% of itssaturation amount; carbon dioxide in an amount that is between about0.04% and about 35% by volume; oxygen in an amount that is between about1% and about 20% by volume; nitrogen in an amount that is greater thanabout 50% by volume; argon in an amount that is greater than about 1.0%by volume; ethanol in an amount that is more than about 1% by volume; 1part per million or less of C₂-C₅ alkynes; 1 part per million or less ofcyclopentadiene; 1 part per million or less of piperylene; and 1 partper million or less of 1,4-pentadiene.

In certain other embodiments, another gaseous isoprene composition isprovided. This composition comprises:

-   -   a. isoprene in an amount between about 0.1% and 15% by volume;    -   b. carbon dioxide in an amount between about 1% and 35% by        volume; and,    -   c. water in an amount that is greater that about 70% of its        saturation amount and wherein the gaseous isoprene composition        comprises 1 part per million or less of C₂-C₅ alkynes. In other        embodiments, the gaseous isoprene composition comprises 1 part        per million or less of each of C₂-C₅ alkynes, cyclopentadiene,        piperylene, and 1,4-pentadiene. In still other embodiments, the        gaseous isoprene composition comprises saturated water. In yet        other embodiments, the gaseous isoprene composition further        comprises oxygen in an amount between about 8% and about 15% by        volume or nitrogen in an amount between about 50% and 75% by        volume or both.

The temperature of the above described gaseous compositions is at least30° C. In some cases, the temperature is between about 30° C. and about60° C. In other cases, the temperature is between about 30° C. and about38° C.

The pressure of the above described gaseous compositions is betweenabout 1 and about 2.5 atmospheres.

For some of the above described gaseous compositions, the temperature isbetween about 30° C. and about 35° C. and is at a pressure between about1 and about 2.5 atmospheres.

Microbially Derived Liquid Isoprene Compositions

Using the methods described herein, the gaseous isoprene compositions ofthe present invention can be further purified to liquid isoprene. Thusin another aspect of the invention, a liquid isoprene composition isprovided that results from the inventive methods. The resulting liquidisoprene composition comprises at least 65% isoprene by weight andwherein the liquid isoprene composition comprises 1 part per million orless of C₂-C₅ alkynes, cyclopentadiene, piperylene, and 1,4-pentadiene.

In some embodiments, the liquid isoprene composition comprises at leastabout 70%, 75%, 80%, 85%, and 90% isoprene by weight. In otherembodiments, the liquid isoprene comprises at least about 95%, 96%, 97%,98%, 99% and 99.5% isoprene by weight. In still other embodiments, theliquid isoprene composition comprises isoprene in an amount that isgreater than about 99.5% by weight.

In other embodiments, the liquid isoprene composition further comprisescarbon dioxide. In some embodiments, the carbon dioxide is present in anamount that is between about 0.01% by weight and about 1% by weight. Inother embodiments, the carbon dioxide is present in an amount that isbetween about 0.05% and about 1% by weight. In further embodiments, thecarbon dioxide is present in an amount that is between about 0.1% andabout 1% by weight. In still further embodiments, the carbon dioxide ispresent in an amount that is between about 0.2% and about 0.7% byweight.

In other embodiments, the liquid isoprene composition further comprisesnitrogen. In some embodiments, the nitrogen is present in an amount thatis between about 0.001% by weight and about 1% by weight. In otherembodiments, the carbon dioxide is present in an amount that is betweenabout 0.01% and about 0.5% by weight. In further embodiments, the carbondioxide is present in an amount that is between about 0.05% and about0.5% by weight.

In other embodiments, the liquid isoprene composition further comprisesethanol. In some embodiments, the ethanol is present in an amount thatis greater than about 0.01% by weight. In other embodiments, the ethanolis present in an amount that is greater than about 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, and 0.9% by weight. In furtherembodiments, the ethanol is present in an amount that is greater thanabout 1% by weight.

In other embodiments, the liquid isoprene composition may comprise waterin an amount that is less than about 1%, 0.5%, 0.1%, and 0.05% byweight. In other embodiments, the liquid isoprene composition maycomprise water in an amount that is less than about 500 ppm, 250 ppm,100 ppm, and 50 ppm by weight. In other embodiments, the liquid isoprenecomposition may comprise water in an amount, by weight, that is lessthan the level of detection.

In other embodiments, the microbial-derived liquid isoprene compositionmay comprise: isoprene in an amount of at least about 65% to an amountgreater than about 99.5% by weight; carbon dioxide in an amount that isbetween about 0.01% and about 1% by weight; nitrogen in an amount thatis between about 0.001% and about 1% by weight; ethanol in an amountgreater than about 0.01% to an amount greater than about 1% by weight;water in an amount that is less than about 1% by weight to an amountthat is less than the level of detection; C₂-C₅ alkynes in an amount 1part per million or less; cyclopentadiene in an amount 1 part permillion or less; piperylene in an amount 1 part per million or less; and1,4-pentadiene in an amount 1 part per million or less.

In certain other embodiments, another liquid isoprene composition isprovided. This composition comprises:

-   -   a. isoprene in an amount greater than about 65% by weight;    -   b. ethanol in an amount greater than about 0.01% by weight; and,    -   c. carbon dioxide in an amount between about 0.01% and about 1%        by weight        wherein the liquid isoprene composition comprises 1 part per        million or less of C₂-C₅ alkynes, cyclopentadiene and piperylene        C₂-C₅ alkynes. In some embodiments, the isoprene is present in        an amount greater than about 85% by weight. In still other        embodiments, the isoprene is present in an amount greater than        about 90% by weight. In further embodiments, the isoprene is        present in an amount greater than about 90% by weight and        ethanol is present in an amount that is between about 0.01% and        about 1% by weight.

For some of the above described liquid isoprene compositions, thecompositions have a temperature below −35° C. and a pressure between0.01 and about 2 atmospheres. In other embodiments, the compositionshave a temperature between −45° C. and about −85° C. and a pressurebelow about 1 atmosphere. In still further embodiments, the compositionshave a temperature below −45° C. and a pressure below about 0.5atmosphere.

Microbial Host Cells

Any microbial host cells capable of making isoprene can be used in themethods herein which would result in the inventive isoprenecompositions.

Illustrative examples of suitable host cells are microbes that have beenshown to make isoprene naturally. These strains include those describedby U.S. Pat. No. 5,849,970 and include: Bacillus amyloliquiefaciens;Bacillus cereus; Bacillus subtillis 6051; Basillus substillis 23059;Bacillus subtillis 23856; Micrococcus luteus; Rhococcus rhodochrous;Acinetobacter calcoacetiucus; Agrobacternum rhizogenes; Escherichiacoli; Erwinia herbicola; Pseudomonoas aeruginosa; and Psuedomonascitronellolis. However, microbes that make isoprene naturally areproduced at extremely low levels.

Isoprene is made from isopentenyl pyrophosphate (IPP) by isoprenesynthase. Because all microbial host cells are capable of making IPP,any host cells can be made to make isoprene by the insertion of isoprenesynthase into its genome. Illustrative examples of suitable nucleotidesequences include but are not limited to: (EF638224, Populus alba);(AJ294819, Populus alba×Populus tremula); (AM410988, Populus nigra);(AY341431, Populus tremuloides); (EF147555, Populus trichocarpa); and(AY316691, Pueraria montana var. lobata). The addition of a heterologousisoprene synthase to a microbial host cells that make isoprene naturallywill improve isoprene yields of natural isoprene producers as well.

Any suitable microbial host cell can be genetically modified to makeisoprene. A genetically modified host cell is one in which nucleic acidmolecules have been inserted, deleted or modified (i.e., mutated; e.g.,by insertion, deletion, substitution, and/or inversion of nucleotides),to produce isoprene. Illustrative examples of suitable host cellsinclude any archae, bacterial, or eukaryotic cell. Examples of archaecells include, but are not limited to those belonging to the genera:Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Methanobacterium,Pyrococcus, Sulfolobus, and Thermoplasma. Illustrative examples ofarchae species include but are not limited to: Aeropyrum pernix,Archaeoglobus fulgidus, Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Pyrococcus abyssi, Pyrococcus horikoshii,Thermoplasma acidophilum, Thermoplasma volcanium.

Examples of bacterial cells include, but are not limited to thosebelonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena,Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium,Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia,Escherichia, Lactobacillus, Lactococcus, Mesorhizobium,Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter,Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun,Serratia, Shigella, Staphylococcus, Strepromyces, Synnecoccus, andZymomonas.

Illustrative examples of bacterial species include but are not limitedto: Bacillus subtilis, Bacillus amyloliquefacines, Brevibacteriumammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii,Enterobacter sakazakii, Escherichia coli, Lactococcus lactis,Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonii,Pseudomonas pudica, Rhodobacter capsulatus, Rhodobacter sphaeroides,Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonellatyphimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,Staphylococcus aureus, and the like.

In general, if a bacterial host cell is used, a non-pathogenic strain ispreferred. Illustrative examples of species with non-pathogenic strainsinclude but are not limited to: Bacillus subtilis, Escherichia coli,Lactibacillus acidophilus, Lactobacillus helveticus, Pseudomonasaeruginosa, Pseudomonas mevalonii, Pseudomonas pudita, Rhodobactersphaeroides, Rodobacter capsulatus, Rhodospirillum rubrum, and the like.

Examples of eukaryotic cells include but are not limited to fungalcells. Examples of fungal cells include, but are not limited to thosebelonging to the genera: Aspergillus, Candida, Chrysosporium,Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora,Penicillium, Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces(formerly Phaffia).

Illustrative examples of eukaryotic species include but are not limitedto: Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Candidaalbicans, Chrysosporium lucknowense, Fusarium graminearum, Fusariumvenenatum, Kluyveromyces lactis, Neurospora crassa, Pichia angusta,Pichia finlandica, Pichia kodamae, Pichia membranaefaciens, Pichiamethanolica, Pichia opuntiae, Pichia pastoris, Pichia pijperi, Pichiaquercuum, Pichia salictaria, Pichia thermotolerans, Pichia trehalophila,Pichia stipitis, Streptomyces ambofaciens, Streptomyces aureofaciens,Streptomyces aureus, Saccaromyces bayanus, Saccaromyces boulardi,Saccharomyces cerevisiae, Streptomyces fungicidicus, Streptomycesgriseochromogenes, Streptomyces griseus, Streptomyces lividans,Streptomyces olivogriseus, Streptomyces rameus, Streptomycestanashiensis, Streptomyces vinaceus, Trichoderma reesei andXanthophyllomyces dendrorhous (formerly Phaffia rhodozyma).

In general, if a eukaryotic cell is used, a non-pathogenic strain ispreferred. Illustrative examples of species with non-pathogenic strainsinclude but are not limited to: Fusarium graminearum, Fusariumvenenatum, Pichia pastoris, Saccaromyces boulardi, and Saccaromycescerevisiae.

In some embodiments, the host cells of the present invention have beendesignated by the Food and Drug Administration as GRAS or GenerallyRegarded As Safe. Illustrative examples of such strains include:Bacillus subtilis, Lactibacillus acidophilus, Lactobacillus helveticus,and Saccharomyces cerevisiae.

In addition to the heterologous nucleic acid encoding an isoprenesynthase, the microbial host cell can be further modified to increaseisoprene yields. These modifications include but are not limited to theexpression of one or more heterologous nucleic acid molecules encodingone or more enzymes in the mevalonate or DXP pathways.

MEV Pathway

A schematic representation of the MEV pathway is described in FIG. 3. Ingeneral, the pathway comprises six steps.

In the first step, two molecules of acetyl-coenzyme A are enzymaticallycombined to form acetoacetyl-CoA. An enzyme known to catalyze this stepis, for example, acetyl-CoA thiolase (also known as acetyl-CoAacetyltransferase). Illustrative examples of nucleotide sequencesinclude but are not limited to the following GenBank accession numbersand the organism from which the sequences derived: (NC_000913 REGION:2324131 . . . 2325315; Escherichia coli), (D49362; Paracoccusdenitrificans), and (L20428; Saccharomyces cerevisiae).

In the second step of the MEV pathway, acetoacetyl-CoA is enzymaticallycondensed with another molecule of acetyl-CoA to form3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An enzyme known to catalyzethis step is, for example, HMG-CoA synthase. Illustrative examples ofnucleotide sequences include but are not limited to: (NC_001145.complement 19061 . . . 20536; Saccharomyces cerevisiae), (X96617;Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907;Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, Locustag SAV2546, GeneID 1122571; Staphylococcus aureus).

In the third step, HMG-CoA is enzymatically converted to mevalonate. Anenzyme known to catalyze this step is, for example, HMG-CoA reductase.Illustrative examples of nucleotide sequences include but are notlimited to: (NM_206548; Drosophila melanogaster), (NC_002758, Locus tagSAV2545, GeneID 1122570; Staphylococcus aureus), (NM_204485; Gallusgallus), (AB015627; Streptomyces sp. KO 3988), (AF542543; Nicotianaattenuata), (AB037907; Kitasatospora griseola), (AX128213, providing thesequence encoding a truncated HMGR; Saccharomyces cerevisiae), and(NC_001145: complement (115734 . . . 118898; Saccharomyces cerevisiae).

In the fourth step, mevalonate is enzymatically phosphorylated to formmevalonate 5-phosphate. An enzyme known to catalyze this step is, forexample, mevalonate kinase. Illustrative examples of nucleotidesequences include but are not limited to: (L77688; Arabidopsisthaliana), and (X55875; Saccharomyces cerevisiae).

In the fifth step, a second phosphate group is enzymatically added tomevalonate 5-phosphate to form mevalonate 5-pyrophosphate. An enzymeknown to catalyze this step is, for example, phosphomevalonate kinase.Illustrative examples of nucleotide sequences include but are notlimited to: (AF429385; Hevea brasiliensis), (NM 006556; Homo sapiens),and (NC 001145. complement 712315 . . . 713670; Saccharomycescerevisiae).

In the sixth step, mevalonate 5-pyrophosphate is enzymatically convertedinto IPP. An enzyme known to catalyze this step is, for example,mevalonate pyrophosphate decarboxylase. Illustrative examples ofnucleotide sequences include but are not limited to: (X97557;Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and(U49260; Homo sapiens).

DXP Pathway

A schematic representation of the DXP pathway is described in FIG. 4. Ingeneral, the DXP pathway comprises seven steps. In the first step,pyruvate is condensed with D-glyceraldehyde 3-phosphate to make1-deoxy-D-xylulose-5-phosphate. An enzyme known to catalyze this stepis, for example, 1-deoxy-D-xylulose-5-phosphate synthase. Illustrativeexamples of nucleotide sequences include but are not limited to:(AF035440; Escherichia coli), (NC_002947, locus tag PP0527; Pseudomonasputida KT2440), (CP000026, locus tag SPA2301; Salmonella entericaParatyphi, see ATCC 9150), (NC_007493, locus tag RSP_0254; Rhodobactersphaeroides 2.4.1), (NC_005296, locus tag RPA0952; Rhodopseudomonaspalustris CGA009), (NC_004556, locus tag PD1293; Xylella fastidiosaTemecula1), and (NC_003076, locus tag AT5G11380; Arabidopsis thaliana).

In the second step, 1-deoxy-D-xylulose-5-phosphate is converted to2C-methyl-D-erythritol-4-phosphate. An enzyme known to catalyze thisstep is, for example, 1-deoxy-D-xylulose-5-phosphate reductoisomerase.Illustrative examples of nucleotide sequences include but are notlimited to: (AB013300; Escherichia coli), (AF148852; Arabidopsisthaliana), (NC_002947, locus tag PP1597; Pseudomonas putida KT2440),(AL939124, locus tag SC05694; Streptomyces coelicolor A3(2)),(NC_007493, locus tag RSP_2709; Rhodobacter sphaeroides 2.4.1), and(NC_007492, locus tag Pfl_1107; Pseudomonas fluorescens PfO-1).

In the third step, 2C-methyl-D-erythritol-4-phosphate is converted to4-diphosphocytidyl-2C-methyl-D-erythritol. An enzyme known to catalyzethis step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritolsynthase. Illustrative examples of nucleotide sequences include but arenot limited to: (AF230736; Escherichia coli), (NC_007493, locus_tagRSP_2835; Rhodobacter sphaeroides 2.4.1), (NC_003071, locus_tagAT2G02500; Arabidopsis thaliana), and (NC_002947, locus_tag PP1614;Pseudomonas putida KT2440).

In the fourth step, 4-diphosphocytidyl-2C-methyl-D-erythritol isconverted to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. Anenzyme known to catalyze this step is, for example,4-diphosphocytidyl-2C-methyl-D-erythritol kinase. Illustrative examplesof nucleotide sequences include but are not limited to: (AF216300;Escherichia coli) and (NC_007493, locus_tag RSP_1779; Rhodobactersphaeroides 2.4.1).

In the fifth step, 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphateis converted to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. An enzymeknown to catalyze this step is, for example, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase. Illustrative examples of nucleotidesequences include but are not limited to: (AF230738; Escherichia coli),(NC_007493, locus_tag RSP_6071; Rhodobacter sphaeroides 2.4.1), and(NC_002947, locus_tag PP1618; Pseudomonas putida KT2440).

In the sixth step, 2C-methyl-D-erythritol 2,4-cyclodiphosphate isconverted to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. An enzymeknown to catalyze this step is, for example,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase. Illustrativeexamples of nucleotide sequences include but are not limited to:(AY033515; Escherichia coli), (NC_002947, locus_tag PP0853; Pseudomonasputida KT2440), and (NC_007493, locus_tag RSP_2982; Rhodobactersphaeroides 2.4.1).

In the seventh step, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate isconverted into either IPP or its isomer, DMAPP. An enzyme known tocatalyze this step is, for example, isopentyl/dimethylallyl diphosphatesynthase. Illustrative examples of nucleotide sequences include but arenot limited to: (AY062212; Escherichia coli) and (NC 002947, locus_tagPP0606; Pseudomonas putida KT2440).

In some embodiments, “cross talk” (or interference) between the hostcell's own metabolic processes and those processes involved with theproduction of IPP as provided by the present invention are minimized oreliminated entirely. For example, cross talk is minimized or eliminatedentirely when the host microorganism relies exclusively on the DXPpathway for synthesizing IPP, and a MEV pathway is introduced to provideadditional IPP. Such host organisms would not be equipped to alter theexpression of the MEV pathway enzymes or process the intermediatesassociated with the MEV pathway. Organisms that rely exclusively orpredominately on the DXP pathway include, for example, Escherichia coli.

In some embodiments, the host cell produces IPP via the MEV pathway,either exclusively or in combination with the DXP pathway. In otherembodiments, a host's DXP pathway is functionally disabled so that thehost cell produces IPP exclusively through a heterologously introducedMEV pathway. The DXP pathway can be functionally disabled by disablinggene expression or inactivating the function of one or more of thenaturally occurring DXP pathway enzymes.

In other embodiments, the host cell produces IPP via the DXP pathway,either exclusively or in combination with the MEV pathway. In otherembodiments, a host's MEV pathway is functionally disabled so that thehost cell produces IPP exclusively through a heterologously introducedDXP pathway. The MEV pathway can be functionally disabled by disablinggene expression or inactivating the function of one or more of thenaturally occurring MEV pathway enzymes.

Methods for genetically modifying host organisms and their cultivationhave been previously described. Illustrative examples include U.S. Pat.Nos. 6,689,593; 7,172,886; 7,183,089; U.S. Patent Publication Nos. US2008/0171378; US 2008/0274523; and US 2009/0203102 and PCT PublicationNos. WO 2007/139924; WO 2009/076676; WO 2010/003007; and WO 2009/132220,which are all incorporated herein by reference in their entirety.Additional methods for modifying host organisms to make isoprene arealso provided in the Examples below.

Purification and Recovery of Microbially Derived Gaseous Isoprene

The present invention provides methods for handling a gaseous isoprenecomposition produced from the microbial host cells. When the resultinggaseous isoprene compositions or the above-described gaseous isoprenecompositions are treated with the following methods, then the resultsare the liquid isoprene compositions described above.

In one aspect, a system for purifying isoprene without extractivedistillation is provided. Extractive distillation is defined asdistillation in the presence of a solvent that forms no azeotrope withother components in the mixture and is used to separate mixtures thatcannot be separated by simple distillation because the volatility of atleast two of the components in the mixture is nearly the same, causingthem to evaporate at nearly the same temperature at a similar rate.Generally miscible, high boiling, and relatively non-volatile, theextraction distillation solvent interacts differently with thecomponents in the mixture enabling the mixture to be separated by normaldistillation. Extractive distillation is almost always used in purifyingpetroleum-derived isoprene. Because extractive distillation requiresspecial equipment and is inherently energy intensive, it is substantialpart of the costs associated with making isoprene. In many embodimentsof the present invention, the resulting isoprene compositions do notinclude trace amounts of an extraction distillation solvent becauseextractive distillation solvents are not used. Illustrative examples ofsuch solvents include but are not limited to acetonitrile anddimethylformamide.

FIG. 1 is a schematic representation of an exemplary separation system.Host cells are cultivated in a bioreactor and the isoprene produced bythe cells vaporizes and forms a gaseous isoprene composition (1).Optionally, the gaseous isoprene composition may pass through a dryingprocess to remove some of the water vapor (not shown). The gaseousisoprene composition (1) is directed to a first chiller 102 which coolsthe gaseous isoprene composition to a temperature between about 10° C.and about −15° C. The cooled gaseous isoprene composition (7) thenpasses through drum 104, where the water vapor in the gaseous isoprenecomposition condenses into a liquid and discharged from the process (5).The exiting gaseous isoprene composition (8) may pass through a dryingprocess to remove any remaining water (not shown). The gaseous isoprenecomposition (8) is directed to a second chiller 106 further cooling thecomposition to a temperature below −35° C. The resulting liquid isoprenecomposition (9) flows to drum 108. Optionally, the bottom stream (10)from drum 108 may then be passed to nitrogen stripper 110 while the topstream (11) is recycled back to the first chiller 102 to assist in (orto serve as the refrigerant for) chilling the incoming gaseous isoprene(1) and then exiting a by-product stream (6). Substantially purenitrogen (2) is introduced into nitrogen stripper 110 whereby the liquidisoprene composition (3) is recovered and the by-product nitrogen gas(4) can be discharged or recovered in a subsequent recovery step (notshown).

In another embodiment, the system comprises:

-   -   a. a bioreactor capable of culturing a plurality of host cells,        preferably the bioreactor has a capacity of greater than 100        liters;    -   b. a first chiller and flash drum operably connected to the        overhead stream of the bioreactor, the first chiller preferably        capable of operating in a temperature range of between 10° C.        and −15° C.;    -   c. a second chiller and flash drum operably connected to the        overhead stream exiting from the first chiller and flash drum,        the second chiller preferably capable of operating in a        temperature below −35° C., for example between about −65° C. and        about −85° C.;    -   d. optionally, the exiting overhead stream from the second        chiller and flash drum may be operably connected to the inlet of        the refrigerant or cooling stream of the first chiller; and    -   e. optionally, the condensed stream exiting from the second        chiller and flash drum may be operably connected to a nitrogen        stripper.

In another aspect, method for recovering isoprene using such a system isprovided. The method comprises:

-   -   a. obtaining a first gaseous composition comprising isoprene and        water wherein the gaseous composition comprises 1 part per        million or less of C₂-C₅ alkynes;    -   b. flowing the first gaseous composition through a first chiller        wherein the first chiller has a temperature of between about        10° C. and about −15° C. thereby resulting in a second gaseous        composition and wherein the second gaseous composition comprises        less water than the first gaseous composition, for example the        second gas composition comprises about 3% by weight or less of        water;    -   c. flowing the second gaseous composition through a second        chiller wherein the second chiller has a temperature below −35°        C.; and    -   d. collecting the resulting liquid isoprene composition.

In other embodiments, the method for recovering isoprene comprisesreducing the water content present in the first gaseous composition byflowing the first gaseous composition through a first chiller whereinthe first chiller has a temperature of between about 10° C. and about−15° C. thereby resulting in a second gaseous composition and whereinthe second gaseous composition comprises less water than the firstgaseous composition, for example the second gas composition comprisesless than about 3% by weight of water. In other embodiments, the secondgas composition comprises less than about 2% by weight of water, lessthan 1%, 0.5%, 0.1%, and 0.05% by weight. In other embodiments, thesecond gas composition comprises less than about 500 ppm by weight ofwater, less than 250 ppm, 100 ppm and 50 ppm by weight.

In another aspect, method for recovering isoprene using such a system isprovided. The method comprises:

-   -   a. obtaining a first gaseous composition comprising isoprene and        water wherein the gaseous composition comprises 1 part per        million or less of C₂-C₅ alkynes;    -   b. flowing the first gaseous composition through a first chiller        wherein the first chiller has a temperature of between about        10° C. and about −15° C. thereby resulting in a second gaseous        composition and wherein the second gaseous composition comprises        less water than the first gaseous composition, for example the        second gas composition comprises about 3% by weight or less of        water;    -   c. flowing the second gaseous composition through a second        chiller wherein the second chiller has a temperature below        −35° C. thereby resulting in a liquid isoprene composition,        wherein the liquid isoprene composition comprises less of one or        more of the following components than the second gaseous        composition, comprising:        -   i. water in amount less than about 1% by weight or less;        -   ii. carbon dioxide in amount less than 1% by weight;        -   iii. nitrogen in amount less than 1% by weight; and    -   d. collecting the resulting liquid isoprene composition.

In some embodiments, the method further comprises nitrogen stripping theliquid isoprene composition. This nitrogen stripping may be accomplishedby any suitable method including passing a substantially pure nitrogenstream through the liquid isoprene enriched composition. This nitrogenstream serves to further remove dissolved gases (such, as for exampleoxygen, carbon dioxide, nitrogen, and argon) and or/remaining water. Inother embodiments, nitrogen stripping of the liquid isoprene compositionmay comprise removing: dissolved oxygen to levels less than about 1%,0.5%, 0.1%, and 0.05% by weight; dissolved carbon dioxide to levels lessthan about 1%, 0.5%, 0.1%, and 0.05% by weight; dissolved nitrogen tolevels less than about 1%, 0.5%, 0.1%, and 0.05% by weight; dissolvedargon to levels less than about 1%, 0.5%, 0.1%, and 0.05% by weight; andany remaining water to levels less than about 1%, 0.5%, 0.1%, 0.05% byweight to levels lower than the level of detection.

In some embodiments, the method further comprises extracting hydrocarbonimpurities at some point in the purifying isoprene process. Thishydrocarbon extraction may be accomplished by passing a portion or allof the first gaseous composition, second gaseous composition and/or theliquid isoprene composition over or through a modified zeolite membraneand/or molecular sieve. The zeolite membrane and/or molecular sieves maybe modified to selectively adsorb either isoprene and not the otherhydrocarbons present in the treated composition or vice versa (adsorbother hydrocarbons in the treated composition and not isoprene). In someembodiments, the zeolites and/or molecular sieves are modified bycarbonization to provide the selected adsorbtivity. In some embodiments,the zeolites may be L-type, Y-type, ZSM-5, and/or beta-type. In someembodiments a method for enhancing the selectivity of a zeolite bycontrolled carbonization as detailed in U.S. Pat. No. 7,041,616, whichis hereby incorporated in its entirety by reference, may be used.

In other embodiments, the first gaseous composition further comprisescarbon dioxide. In still other embodiments, the first gaseouscomposition further comprises oxygen. In still other embodiments, thefirst gaseous composition further comprises nitrogen.

In other embodiments, the first gaseous composition comprises 1 part permillion or less of C₂-C₅ alkynes and cyclopentadiene, In furtherembodiments, the first gaseous composition further comprises 1 part permillion or less of C₂-C₅ alkynes and piperylene. In still otherembodiments, the first gaseous composition comprises 1 part per millionor less of C₂-C₅ alkynes and 1,4-pentadiene. In still other embodiments,the first gaseous composition comprises 1 part per million or less ofC₂-C₅ alkynes, cyclopentadiene, piperylene, and 1,4-pentadiene.

In other embodiments, the first gaseous composition is flowed through adrier prior to flowing through the first chiller. In still otherembodiments, the first gaseous composition is flowed through a drierafter flowing through the first chiller but prior to flowing through thesecond chiller.

In other embodiments, the first chiller has a temperature of about −5°C. In other embodiments, the first chiller is cooled using a propylenerefrigeration system. In still other embodiments, the first chiller iscooled using an ammonium refrigeration system.

In other embodiments, the second chiller has a temperature less thanabout −50° C. In other embodiments, the second chiller has a temperatureof about −60° C. to about −85° C. In still other embodiments, the secondchiller has a temperature of less than about −65° C. In still otherembodiments, the second chiller has a temperature between about −35° C.and about −85° C.

In other embodiments, the liquid isoprene composition comprises at leastabout 70%, 75%, 80%, 85%, and 90% isoprene by weight. In otherembodiments, the liquid isoprene comprises at least about 95%, 96%, 97%,98%, 99% and 99.5% isoprene by weight. In still other embodiments, theliquid isoprene composition comprises isoprene in an amount that isgreater than about 99.5% by weight.

In other embodiments, the second chiller is cooled using an ethylenerefrigeration system.

In another aspect, another method is provided. The method comprises:

-   -   a. culturing a plurality of host cells capable of making        isoprene;    -   b. forming a first gaseous composition comprising isoprene and        water wherein the water is present in an amount greater than        about 70% of its saturation amount;    -   c. subjecting the first gaseous composition to a first cooling        step whereby substantially all of the water is removed from the        first gaseous composition resulting a second gaseous        composition;    -   d. subjecting the second gaseous composition to a second cooling        step whereby a liquid isoprene composition is collected.

In some embodiments, the method further comprises nitrogen stripping theliquid isoprene composition.

In other embodiments, the first gaseous composition further comprisescarbon dioxide. In still other embodiments, the first gaseouscomposition further comprises oxygen. In still other embodiments, thefirst gaseous composition further comprises nitrogen.

In other embodiments, the first gaseous composition comprises 1 part permillion or less of C₂-C₅ alkynes and cyclopentadiene. In furtherembodiments, the first gaseous composition further comprises 1 part permillion or less of C₂-C₅ alkynes and piperylene. In still otherembodiments, the first gaseous composition comprises 1 part per millionor less of C₂-C₅ alkynes and 1,4-pentadiene. In still other embodiments,the first gaseous composition comprises 1 part per million or less ofC₂-C₅ alkynes, cyclopentadiene, piperylene, and 1,4-pentadiene.

In other embodiments, the first gaseous composition is subjected througha drier prior to the first cooling step. In still other embodiments, thefirst gaseous composition is subjected through a drier after the firstcooling step but prior to the second cooling step.

In other embodiments, the first cooling step cools the first gaseousisoprene composition to a temperature between about 10° C. and about−15° C., between about 10° C. and about −10° C., about 5° C. and about−5° C., and about 5° C. and about −10° C.

In other embodiments, the first cooling step uses a propylenerefrigeration system. In still other embodiments, the first cooling stepuses chiller an ammonium refrigeration system.

In other embodiments, the second cooling step cools the second gaseousisoprene composition to a temperature less than −35° C. In otherembodiments, the second cooling step cools the second gaseous isoprenecomposition to a temperature less than about −50° C. In still otherembodiments, the second cooling step cools the second gaseous isoprenecomposition to a temperature about −60° C. to about −85° C. In stillfurther embodiments, the second cooling step cools the second gaseousisoprene composition to a temperature of less than about −65° C.

In other embodiments, the second cooling step uses an ethylenerefrigeration system.

In other embodiments, the liquid isoprene composition comprises at leastabout 70%, 75%, 80%, 85%, and 90% isoprene by weight. In otherembodiments, the liquid isoprene comprises at least about 95%, 96%, 97%,98%, 99% and 99.5% isoprene by weight. In still other embodiments, theliquid isoprene composition comprises isoprene in an amount that isgreater than about 99.5% by weight.

In other embodiments, the host cells are selected from the genusBacillus, Escherichia or Acinetobacter. In still other embodiments, thehost cells are Escherichia coli. In further embodiments, the host cellsare yeast. In still further embodiments, the host cells areSaccharomyces cerevisiae.

In another aspect, another method is provided. The method comprises:

-   -   a. contacting a plurality of host cells capable of making        isoprene in an aqueous medium wherein the aqueous medium is in        contact with an immiscible organic liquid and the aqueous        medium, the host cells, and the immiscible organic liquid is in        a closed vessel; and    -   b. culturing the host cells in the aqueous medium whereby the        host cells make isoprene and the isoprene is captured in the        immiscible organic liquid.

In some embodiments, the method further comprises separating theimmiscible organic liquid from the aqueous medium and separating theisoprene from the immiscible organic liquid.

In other embodiments, the immiscible organic liquid is selected frombutyl acetate, ethyl acetate, isopropyl myristate, methyl isobutylketone, methyl oleate, and toluene. In certain embodiments, the solventis butyl acetate. In other embodiments, the immiscible organic liquid isisopropyl myristrate.

In other embodiments, the isoprene is separated from the immiscibleorganic liquid by heating the immiscible organic liquid to a temperatureabove 34° C.

The resulting gaseous isoprene composition can then be further purifiedusing the methods described above.

In another aspect, a system for making microbial isoprene is provided.An illustrative example of such a system is shown in FIG. 2. Bioreactor202 is a closed system where host cells capable of making isoprene arecultivated in an aqueous medium and with an immiscible organic liquid ontop of the aqueous medium. Because bioreactor 202 is a closed system,the isoprene produced by the host cells is captured by the immiscibleorganic liquid. The isoprene-enriched immiscible organic liquid (A) maythen directed be directed to any suitable gas-liquid separation method.In this example, the isoprene-enriched immiscible organic liquid isdirected to a heater 204 which volatizes the isoprene into a gaseousisoprene composition (1). This gaseous isoprene composition (1) can thenbe further purified using the systems and methods described above. Theimmiscible organic liquid (B) can optionally be recycled and used insubsequent bioreactions to make isoprene.

In another embodiment, the system comprises:

-   -   a. a closed vessel;    -   b. an aqueous medium, within the vessel, forming a first phase;    -   c. a plurality of host cells, within the aqueous medium, capable        of making isoprene; and,    -   d. a liquid organic second phase, capable of capturing the        isoprene made by the host cells, in contact with the first        phase.

In some embodiments, the immiscible organic liquid is selected frombutyl acetate, ethyl acetate, isopropyl myristate, methyl isobutylketone, methyl oleate, and toluene. In certain embodiments, the solventis butyl acetate. In other embodiments, the immiscible organic liquid isisopropyl myristrate.

In other embodiments, the host cells are selected from the genusBacillus, Escherichia or Acinetobacter. In still other embodiments, thehost cells are Escherichia coli. In further embodiments, the host cellsare yeast. In still further embodiments, the host cells areSaccharomyces cerevisiae.

EXAMPLES

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of the biosynthetic industry and thelike, which are within the skill of the art. To the extent suchtechniques are not described fully herein, one can find ample referenceto them in the scientific literature.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (for example, amounts, temperature, and soon), but variation and deviation can be accommodated, and in the event aclerical error in the numbers reported herein exists, one of ordinaryskill in the arts to which this invention pertains can deduce thecorrect amount in view of the remaining disclosure herein. Unlessindicated otherwise, temperature is reported in degrees Celsius, andpressure is at or near atmospheric pressure at sea level. All reagents,unless otherwise indicated, were obtained commercially. The followingexamples are intended for illustrative purposes only and do not limit inany way the scope of the present invention.

Example 1

This example describes methods for detecting isoprene trapped in animmiscible organic liquid in a closed fermentation system.

In this example, isopropyl myristate (IPM) and solutions of IPM spikedwith 103 mg/L, 10.3 mg/L and 0 mg/L isoprene were prepared and stored in100 ml capped media bottles.

125 ml unbaffled flasks with screw caps/septa were set up in triplicate.The flasks contained 40 ml medium (Yeast Nitrogen Base media with 4%galactose, 0.2% glucose, Leu) inoculated with overnight yeast culture(which does not make isoprene) grown to an OD=0.05 with 8 ml of IPMsolutions with the various concentrations of isoprene. The sealed flaskswere incubated for 72 hours at 30° C. and 200 rpm.

Post-incubation, the IPM overlay was phase-separated by manual transferto primary GC vials, then transferred to secondary GC vials and runundiluted on GC-FID. In addition to samples from flasks, the originalsolutions of 103 and 10.3 mg/L isoprene in IPM (NOT shaken for 72 hoursat 200 rpm, 30° C.) were also analyzed.

As shown by FIG. 5, over 90% of the spiked isoprene was recovered fromthe IPM layer. Cell growth for cultures with 103, 10.3, and 0 mg/Lisoprene in the IPM layer was indistinguishable.

Example 2

This example describes methods for making nucleic acids for expressingin Saccharomyces cerevisiae heterologous isoprene synthases.

Genomic DNA was isolated from Saccharomyces cerevisiae strains Y002(CEN.PK2 background MATA ura3-52 trp1-289 leu2-3,112 his3Δ1 MAL2-8CSUC2) (van Dijken et al. (2000) Enzyme and Microbial Technology26:706-714), Y007 (S288C background MATA trp1Δ63) (ATCC number 200873),Y051 (S288C background), and EG123 (ATCC number 204278). The strainswere grown overnight in liquid medium containing 1% Yeast extract, 2%Bacto-peptone, and 2% Dextrose (YPD medium). Cells were isolated from 10mL liquid cultures by centrifugation at 3,100 rpm, washing of cellpellets in 10 mL ultra-pure water, and re-centrifugation. Genomic DNAwas extracted using the Y-DER yeast DNA extraction kit (PierceBiotechnologies, Rockford, Ill.) as per manufacturer's suggestedprotocol. Extracted genomic DNA was re-suspended in 100 uL 10 mMTris-Cl, pH 8.5, and OD_(260/280) readings were taken on a ND-1000spectrophotometer (NanoDrop Technologies, Wilmington, Del.) to determinegenomic DNA concentration and purity.

DNA amplification by Polymerase Chain Reaction (PCR) was done in anApplied Biosystems 2720 Thermocycler (Applied Biosystems Inc, FosterCity, Calif.) using the Phusion High Fidelity DNA Polymerase system(Finnzymes OY, Espoo, Finland) as per manufacturer's suggested protocol.Upon the completion of a PCR amplification of a DNA fragment that was tobe inserted into the pCR® 4Blunt-TOPO vector (Invitrogen, Carlsbad,Calif.), A nucleotide overhangs were created by adding 1 uL of QiagenTaq Polymerase (Qiagen, Valencia, Calif.) to the reaction mixture andperforming an additional 10 minute, 72° C. PCR extension step, followedby cooling to 4° C. Upon completion of a PCR amplification, 8 uL of a50% glycerol solution was added to the reaction mix.

Agarose gel electrophoresis was performed using a 1% TBE (0.89 M Tris,0.89 M Boric acid, 0.02 M EDTA sodium salt) agarose gel containing 0.5μg/mL ethidium bromide, at 120 V, 400 mA for 30 minutes. DNA bands werevisualized using ultraviolet light. DNA bands were excised from the gelwith a sterile razor blade, and the excised DNA was gel purified usingthe Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, Calif.)according to manufacturer's suggested protocols. The purified DNA waseluted into 10 uL ultra-pure water, and OD_(260/280) readings were takenon a ND-1000 spectrophotometer to determine DNA concentration andpurity.

Ligations were performed using 100-500 μg of purified PCR product andHigh Concentration T4 DNA Ligase (New England Biolabs, Ipswich, Mass.)as per manufacturer's suggested protocol. For plasmid propagation,ligated constructs were transformed into Escherichia coli DH5achemically competent cells (Invitrogen, Carlsbad, Calif.) as permanufacturer's suggested protocol. Positive transformants were selectedon solid media containing 1.5% Bacto Agar, 1% Tryptone, 0.5% YeastExtract, 1% NaCl, and an appropriate antibiotic. Isolated transformantswere grown for 16 hours in liquid LB medium containing 50 μg/mLcarbenicillin or kanamycin antibiotic at 37° C., and plasmid wasisolated and purified using a QIAprep Spin Miniprep kit (Qiagen,Valencia, Calif.) as per manufacturer's suggested protocol. Constructswere verified by performing diagnostic restriction enzyme digestions,resolving DNA fragments on an agarose gel, and visualizing the bandsusing ultraviolet light. Select constructs were also verified by DNAsequencing, which was done by Elim Biopharmaceuticals Inc. (Hayward,Calif.).

Expression plasmid pAM353 was generated by inserting a nucleotidesequence encoding a β-farnesene synthase into the pRS425-Gall vector(Mumberg et. al. (1994) Nucl. Acids. Res. 22(25): 5767-5768). Thenucleotide sequence insert was generated synthetically, using as atemplate the coding sequence of the β-farnesene synthase gene ofArtemisia annua (GenBank accession number AY835398) codon-optimized forexpression in Saccharomyces cerevisiae (SEQ ID NO: 1). The syntheticallygenerated nucleotide sequence was flanked by 5′ BamHI and 3′ Xho1restriction sites, and could thus be cloned into compatible restrictionsites of a cloning vector such as a standard pUC or pACYC origin vector.The synthetically generated nucleotide sequence was isolated bydigesting to completion the construct using BamHI and Xho1 restrictionendonucleases. The reaction mixture was resolved by gel electrophoresis,the approximately 1.7 kb DNA fragment comprising the β-farnesenesynthase coding sequence was gel purified, and the isolated DNA fragmentwas ligated into the BamHI Xho1 restriction site of the pRS425-Gallvector, yielding expression plasmid pAM353.

Expression plasmid pAM404 was generated by inserting a nucleotidesequence encoding the β-farnesene synthase of Artemisia annua (GenBankaccession number AY835398) codon-optimized for expression inSaccharomyces cerevisiae into vector pAM178 (SEQ ID NO: 2). Thenucleotide sequence encoding the β-farnesene synthase was PCR amplifiedfrom pAM353 using primers 52-84 pAM326 BamHI (SEQ ID NO: 21) and 52-84pAM326 NheI (SEQ ID NO: 22). The resulting PCR product was digested tocompletion using BamHI and NheI restriction endonucleases, the reactionmixture was resolved by gel electrophoresis, the approximately 1.7 kbDNA fragment comprising the β-farnesene synthase coding sequence was gelpurified, and the isolated DNA fragment was ligated into the BamHI NheIrestriction site of vector pAM178, yielding expression plasmid pAM404.

Plasmid Genetrix2080 was generated by inserting a nucleotide sequenceencoding an isoprene synthase into vector pUC19. The insert wasgenerated synthetically as two approximately equal sized DNA fragments,fragment 2080_1 (SEQ ID NO: 3) and fragment 2080_2 (SEQ ID NO: 4), usingas a template the coding sequence of the isoprene synthase gene of Kudzucodon-optimized for expression in Saccharomyces cerevisiae. Each DNAfragment was flanked by LguI restriction sites, and comprised a 40 basepair overlapping sequence at one end. The synthetically generated DNAfragments were blunt ligated into the SmaI restriction site of the pUC19cloning vector, from which the two inserts were excised again bydigesting to completion 500 μg of the construct using LguI restrictionendonuclease (Fermentas, Glen Burnie, Md.). The restriction endonucleasewas heat inactivated for 20 minutes at 65° C., and the DNA fragmentswere stitched together by a first round of PCR amplification (one cycleof denature at 98° C. for 2 minutes; 5 cycles of denature at 98° C. for30 seconds and anneal/extend at 72° C. for 30 seconds per kilobase PCRproduct; no primers were used). Samples were placed on ice, 0.5 uM ofeach terminal primer TRIX_L_494 (SEQ ID NO: 79) and TRIX_L_495 (SEQ IDNO: 80) were added to the reaction mixture, and a second round of PCRamplification was performed (one cycle of denature at 98° C. for 2minutes; 35 rounds of denature at 98° C. for 12 seconds andanneal/extend at 72° C. for 20 seconds per kilobase PCR product; onecycle of final extend at 72° C. for 7 minutes; and a final hold at 4°C.). The reaction mixture was resolved by gel electrophoresis, theassembled DNA fragment was gel purified, treated with T4 polynucleotidekinase (PNK) (New England Biolabs, Ipswich, Mass.), and blunt ligatedinto the SmaI restriction site of vector pUC19, yielding plasmidGenetrix2080.

Plasmid Genetrix2081 was generated by inserting a nucleotide sequenceencoding an isoprene synthase into vector pUC19. The insert wasgenerated synthetically as two approximately equal sized DNA fragments,fragment 2081_1 (SEQ ID NO: 5) and fragment 2081_2 (SEQ ID NO: 6), usingas a template the coding sequence of the isoprene synthase gene ofPopulus nigra codon-optimized for expression in Saccharomycescerevisiae. Each DNA fragment was flanked by LguI restriction sites, andcomprised a 30 base pair overlapping sequence at one end. Thesynthetically generated DNA fragments were blunt ligated into the SmaIrestriction site of the pUC19 cloning vector, from which the two insertswere excised again by digesting to completion 500 μg of the constructusing LguI restriction endonuclease (Fermentas, Glen Burnie, Md.). Therestriction endonuclease was heat inactivated for 20 minutes at 65° C.,and the DNA fragments were stitched together by a first round of PCRamplification (one cycle of denature at 98° C. for 2 minutes; 5 cyclesof denature at 98° C. for 30 seconds and anneal/extend at 72° C. for 30seconds per kilobase PCR product; no primers were used). Samples wereplaced on ice, 0.5 uM of each terminal primer TRIX_L_497 (SEQ ID NO: 81)and TRIX_L498 (SEQ ID NO: 82) were added to the reaction mixture, and asecond round of PCR amplification was performed (one cycle of denatureat 98° C. for 2 minutes; 35 rounds of denature at 98° C. for 12 secondsand anneal/extend at 72° C. for 20 seconds per kilobase PCR product; onecycle of final extend at 72° C. for 7 minutes; and a final hold at 4°C.). The reaction mixture was resolved by gel electrophoresis, theassembled DNA fragment was gel purified, treated with T4 polynucleotidekinase (PNK) (New England Biolabs, Ipswich, Mass.), and blunt ligatedinto the SmaI restriction site of vector pUC19, yielding plasmidGenetrix2081.

Plasmid Genetrix2082 was generated by inserting a nucleotide sequenceencoding an isoprene synthase into vector pUC19. The insert wasgenerated synthetically as two approximately equal sized DNA fragments,fragment 2082_1 (SEQ ID NO: 7) and fragment 2082_2 (SEQ ID NO: 8), usingas a template the coding sequence of the isoprene synthase gene ofPopulus alba×Populus tremula codon-optimized for expression inSaccharomyces cerevisiae. Each DNA fragment was flanked by LguIrestriction sites, and comprised a 40 base pair overlapping sequence atone end. The synthetically generated DNA fragments were blunt ligatedinto the SmaI restriction site of the pUC19 cloning vector, from whichthe two inserts were excised again by digesting to completion 500 μg ofthe construct using LguI restriction endonuclease (Fermentas, GlenBurnie, Md.). The restriction endonuclease was heat inactivated for 20minutes at 65° C., and the DNA fragments were stitched together by afirst round of PCR amplification (one cycle of denature at 98° C. for 2minutes; 5 cycles of denature at 98° C. for 30 seconds and anneal/extendat 72° C. for 30 seconds per kilobase PCR product; no primers wereused). Samples were placed on ice, 0.5 uM of each terminal primerTRIXL_500 (SEQ ID NO: 83) and TRIX_L_501 (SEQ ID NO: 84) were added tothe reaction mixture, and a second round of PCR amplification wasperformed (one cycle of denature at 98° C. for 2 minutes; 35 rounds ofdenature at 98° C. for 12 seconds and anneal/extend at 72° C. for 20seconds per kilobase PCR product; one cycle of final extend at 72° C.for 7 minutes; and a final hold at 4° C.). The reaction mixture wasresolved by gel electrophoresis, the assembled DNA fragment was gelpurified, treated with T4 polynucleotide kinase (PNK) (New EnglandBiolabs, Ipswich, Mass.), and blunt ligated into the SmaI restrictionsite of vector pUC19, yielding plasmid Genetrix2082.

Expression plasmid pAM1547 was generated by replacing the β-farnesenesynthase coding sequence of expression plasmid pAM404 with the isoprenesynthase coding sequence of plasmid Genetrix2080. DNA fragment IS2080was generated by PCR amplifying plasmid Genetrix2080 using primersYD-198-70A (SEQ ID NO: 85) and YD-198-70B (SEQ ID NO: 86), digesting thePCR product to completion using restriction endonucleases BamHI andNheI, resolving the reaction mixture by gel electrophoresis, and gelpurifying the approximately 1.8 kb DNA fragment comprising the isoprenesynthase coding sequence. Expression plasmid pAM404 was digested tocompletion using restriction endonucleases BamHI and NheI, the reactionmixture was resolved by gel electrophoresis, the approximately 7.3 kbvector backbone (lacking the β-farnesene synthase coding sequence) wasgel purified, and the purified vector backbone was ligated with DNAfragment IS2080, yielding pAM1547 (see FIG. 6 for a plasmid map).

Expression plasmid pAM1548 was generated by replacing the β-farnesenesynthase coding sequence of expression plasmid pAM404 with a truncatedversion of the isoprene synthase coding sequence of plasmidGenetrix2080. DNA fragment IS2080T was generated by PCR amplifyingplasmid Genetrix2080 using primers YD-198-70B (SEQ ID NO: 86) andYD-198-70G (SEQ ID NO: 91), digesting the PCR product to completionusing restriction endonucleases BamHI and NheI, resolving the reactionmixture by gel electrophoresis, and gel purifying the approximately 1.65kb DNA fragment comprising the truncated isoprene synthase codingsequence. pAM404 was digested to completion using restrictionendonucleases BamHI and NheI, the reaction mixture was resolved by gelelectrophoresis, the approximately 7.3 kb backbone (lacking theβ-farnesene synthase coding sequence) was gel purified, and the purifiedvector backbone was ligated with the amplified DNA fragment IS2080T,yielding pAM1548.

Expression plasmid pAM1549 was generated by replacing the β-farnesenesynthase coding sequence of expression plasmid pAM404 with the isoprenesynthase coding sequence of plasmid Genetrix2081. DNA fragment IS2081was generated by PCR amplifying plasmid Genetrix2081 using primersYD-198-70C (SEQ ID NO: 87) and YD-198-70D (SEQ ID NO: 88), digesting thePCR product to completion using restriction endonucleases BamHI andNheI, resolving the reaction mixture by gel electrophoresis, and gelpurifying the approximately 1.8 kb DNA fragment comprising the isoprenesynthase coding sequence. Expression plasmid pAM404 was digested tocompletion using restriction endonucleases BamHI and NheI, the reactionmixture was resolved by gel electrophoresis, the approximately 7.3 kbvector backbone (lacking the β-farnesene synthase coding sequence) wasgel purified, and the purified vector backbone was ligated with DNAfragment IS2081, yielding pAM1549.

Expression plasmid pAM1550 was generated by replacing the β-farnesenesynthase coding sequence of expression plasmid pAM404 with a truncatedversion of the isoprene synthase coding sequence of plasmidGenetrix2081. DNA fragment IS2081T was generated by PCR amplifyingplasmid Genetrix2081 using primers YD-198-70D (SEQ ID NO: 88) andYD-198-70H (SEQ ID NO: 92), digesting the PCR product to completionusing restriction endonucleases BamHI and NheI, resolving the reactionmixture by gel electrophoresis, and gel purifying the approximately 1.6kb DNA fragment comprising the truncated isoprene synthase codingsequence. Expression plasmid pAM404 was digested to completion usingrestriction endonucleases BamHI and NheI, the reaction mixture wasresolved by gel electrophoresis, the approximately 7.3 kb vectorbackbone (lacking the (3-farnesene synthase coding sequence) was gelpurified, and the purified vector backbone was ligated with DNA fragmentIS2081T, yielding pAM1550.

Expression plasmid pAM1551 was generated by replacing the β-farnesenesynthase coding sequence of expression plasmid pAM404 with the isoprenesynthase coding sequence of plasmid Genetrix2082. DNA fragment IS2082was generated by PCR amplifying plasmid Genetrix2082 using primersYD-198-70E (SEQ ID NO: 89) and YD-198-70F (SEQ ID NO: 90), digesting thePCR product to completion using restriction endonucleases BamHI andNheI, resolving the reaction mixture by gel electrophoresis, and gelpurifying the approximately 1.8 kb DNA fragment comprising the isoprenesynthase coding sequence. Expression plasmid pAM404 was digested tocompletion using restriction endonucleases BamHI and NheI, the reactionmixture was resolved by gel electrophoresis, the approximately 7.3 kbvector backbone (lacking the β-farnesene synthase coding sequence) wasgel purified, and the purified vector backbone was ligated with DNAfragment IS2082, yielding pAM1551.

Expression plasmid pAM1552 was generated by replacing the β-farnesenesynthase coding sequence of expression plasmid pAM404 with a truncatedversion of the isoprene synthase coding sequence of plasmidGenetrix2082. DNA fragment IS2082T was generated by PCR amplifyingplasmid Genetrix2082 using primers YD-198-70F (SEQ ID NO: 90) andYD-198-70I (SEQ ID NO: 93), digesting the PCR product to completionusing restriction endonucleases BamHI and NheI, resolving the reactionmixture by gel electrophoresis, and gel purifying the approximately 1.64kb DNA fragment comprising the truncated isoprene synthase codingsequence. Expression plasmid pAM404 was digested to completion usingrestriction endonucleases BamHI and NheI, the reaction mixture wasresolved by gel electrophoresis, the approximately 7.3 kb vectorbackbone (lacking the β-farnesene synthase coding sequence) was gelpurified, and the purified vector backbone was ligated with DNA fragmentIS2082T, yielding pAM1552.

Plasmid pAM840 was generated by inserting the coding sequence of thehisG gene into the pCR® 2.1-TOPO vector (Invitrogen, Carlsbad, Calif.).The coding sequence of the hisG gene was PCR amplified using primersKB34 (SEQ ID NO: 75) and KB39 (SEQ ID NO: 76) and plasmid pNKY51 (Alaniet al. (1987) Genetics 116(4):541-555) as template. The amplified DNAfragment was ligated with the Topo vector as per manufacturer'ssuggested protocol, yielding pAM840.

Plasmid pAM728 was generated by introducing the coding sequence of thefarnesene synthase gene of Artemisia annua (GenBank accession numberAY835398) codon-optimized for expression in Saccharomyces cerevisiae andunder control of the promoter of the GAL7 gene of Sacharomycescerevisiae (P_(GAL7)) into plasmid pRS425 (Christianson et al. (1992)Gene 110(1):119-122). An approximately 0.5 kb DNA fragment comprisingP_(GAL7) was PCR amplified from Y002 genomic DNA using primersGW-110-26-pGAL7-PstI F (SEQ ID NO: 119) and GW-110-26-pGAL7 R (SEQ IDNO: 120) and was gel purified. An approximately 2 kb DNA fragmentcomprising the coding sequence of the farnesene synthase gene was PCRamplified using primers GW-110-26-pGAL7-FS F (SEQ ID NO: 121) andGW-110-26-FS-BamHI R (SEQ ID NO: 122). The two DNA fragments werestitched together using PCR primers GW-110-26-pGAL7-PstI F (SEQ ID NO:119) and GW-110-26-FS-BamHI R (SEQ ID NO: 122) to create aP_(GAL7)-FS-tCYC1 insert. The P_(GAL7)-FS-tCYC1 insert and plasmidpRS425 were digested to completion using PstI and BamHI restrictionendonucleases, and the two DNA fragments were ligated, yielding pAM728.

Plasmid pAM940 was generated by introducing the farnesene synthasesequence of plasmid pAM728 into plasmid pRS426 (Christianson et al.(1992) Gene 110(1):119-122). Plasmids pAM728 and pRS426 were digested tocompletion using Xho1 and BamHI restriction endonucleases, the reactionmixtures were resolved by gel electrophoresis, the approximately 5.7 kbpRS426 vector backbone and the approximately 2.5 kb P_(GAL7)-FS-tCYC1insert of pAM728 were gel purified, and the two DNA fragments wereligated, yielding plasmid pAM940.

Plasmid pAM489 was generated by inserting the ERG20-P_(GAL)-tHMGR insertof vector pAM471 into vector pAM466. Vector pAM471 was generated byinserting DNA fragment ERG20-P_(GAL)-tHMGR, which comprises the openreading frame (ORF) of the ERG20 gene of Saccharomyces cerevisiae (ERG20nucleotide positions 1 to 1208; A of ATG start codon is nucleotide 1)(ERG20), the genomic locus containing the divergent GAL1 and GAL10promoter of Saccharomyces cerevisiae (GAL1 nucleotide position −1 to−668) (P_(GAL)), and a truncated ORF of the HMG1 gene of Saccharomycescerevisiae (HMG1 nucleotide positions 1586 to 3323) (tHMGR), into theTOPO Zero Blunt II cloning vector (Invitrogen, Carlsbad, Calif.). VectorpAM466 was generated by inserting DNA fragment TRP1^(−856 to +548),which comprises a segment of the wild-type TRP1 locus of Saccharomycescerevisiae that extends from nucleotide position −856 to position 548and harbors a non-native internal XmaI restriction site between bases−226 and −225, into the TOPO TA pCR2.1 cloning vector (Invitrogen, qCarlsbad, Calif.). DNA fragments ERG20-P_(GAL)-tHMGR andTRP1^(−856 to +548) were generated by PCR amplification as outlined inTable 1. For the construction of pAM489, 400 ng of pAM471 and 100 ng ofpAM466 were digested to completion using XmaI restriction enzyme (NewEngland Biolabs, Ipswich, Mass.), DNA fragments corresponding to theERG20-P_(GAL)-tHMGR insert and the linearized pAM466 vector were gelpurified, and 4 molar equivalents of the purified insert was ligatedwith 1 molar equivalent of the purified linearized vector, yieldingpAM489. FIG. 7A shows a map of the ERG20-P_(GAL)-tHMGR insert, and SEQID NO: 9 shows the nucleotide sequence of the DNA fragment and theflanking TRP1 sequences.

TABLE 1 PCR amplifications performed to generate pAM489 PCR RoundTemplate Primer 1 Primer 2 PCR Product 1 100 ng of Y051 genomic61-67-CPK001-G 61-67-CPK002-G TRP1^(−856 to −226) DNA (SEQ ID NO: 23)(SEQ ID NO: 24) 61-67-CPK003-G 61-67-CPK004-G TRP1^(−225-to +548) (SEQID NO: 25) (SEQ ID NO: 26) 100 ng of EG123 genomic 61-67-CPK025-G61-67-CPK050-G ERG20 DNA (SEQ ID NO: 47) (SEQ ID NO: 55) 100 ng of Y002genomic 61-67-CPK051-G 61-67-CPK052-G P_(GAL) DNA (SEQ ID NO: 56) (SEQID NO: 57) 61-67-CPK053-G 61-67-CPK031-G tHMGR (SEQ ID NO: 58) (SEQ IDNO: 48) 2 100 ng each of TRP1^(−856 to −226) 61-67-CPK001-G61-67-CPK004-G TRP1^(−856 to +548) and TRP1^(−225-to +548) purified (SEQID NO: 23) (SEQ ID NO: 26) PCR products 100 ng each of ERG20 and61-67-CPK025-G 61-67-CPK052-G ERG20-P_(GAL) P_(GAL) purified PCRproducts (SEQ ID NO: 47) (SEQ ID NO: 57) 3 100 ng each of ERG20-P_(GAL)61-67-CPK025-G 61-67-CPK031-G ERG20-P_(GAL)- and tHMGR purified PCR (SEQID NO: 47) (SEQ ID NO: 48) tHMGR products

Plasmid pAM491 was generated by inserting the ERG13-P_(GAL)-tHMGR insertof vector pAM472 into vector pAM467. Vector pAM472 was generated byinserting DNA fragment ERG13-P_(GAL)-tHMGR, which comprises the ORF ofthe ERG13 gene of Saccharomyces cerevisiae (ERG13 nucleotide positions 1to 1626) (ERG13), the genomic locus containing the divergent GAL1 andGAL10 promoter of Saccharomyces cerevisiae (GAL1 nucleotide position −1to −668) (P_(GAL)), and a truncated ORF of the HMG1 gene ofSaccharomyces cerevisiae (HMG1 nucleotide position 1586 to 3323)(tHMGR), into the TOPO Zero Blunt II cloning vector. Vector pAM467 wasgenerated by inserting DNA fragment URA3^(−723 to 701), which comprisesa segment of the wild-type URA3 locus of Saccharomyces cerevisiae thatextends from nucleotide position −723 to position −224 and harbors anon-native internal XmaI restriction site between bases −224 and −223,into the TOPO TA pCR2.1 cloning vector. DNA fragmentsERG13-P_(GAL)-tHMGR and URA3^(−723 to 701) were generated by PCRamplification as outlined in Table 2. For the construction of pAM491,400 ng of pAM472 and 100 ng of pAM467 were digested to completion usingXmaI restriction enzyme, DNA fragments corresponding to theERG13-P_(GAL)-tHMGR insert and the linearized pAM467 vector were gelpurified, and 4 molar equivalents of the purified insert was ligatedwith 1 molar equivalent of the purified linearized vector, yieldingpAM491. FIG. 7B shows a map of the ERG13-P_(GAL)-tHMGR insert, and SEQID NO: 10 shows the nucleotide sequence of the DNA fragment and theflanking URA3 sequences.

TABLE 2 PCR amplifications performed to generate pAM491 PCR RoundTemplate Primer 1 Primer 2 PCR Product 1 100 ng of Y007 genomic61-67-CPK005-G 61-67-CPK006-G URA3^(−723 to −224) DNA (SEQ ID NO: 27)(SEQ ID NO: 28) 61-67-CPK007-G 61-67-CPK008-G URA3^(−223 to 701) (SEQ IDNO: 29) (SEQ ID NO: 30) 100 ng of Y002 genomic 61-67-CPK032-G61-67-CPK054-G ERG13 DNA (SEQ ID NO: 49) (SEQ ID NO: 59) 61-67-CPK052-G61-67-CPK055-G P_(GAL) (SEQ ID NO: 57) (SEQ ID NO: 60) 61-67-CPK031-G61-67-CPK053-G tHMGR (SEQ ID NO: 48) (SEQ ID NO: 58) 2 100 ng each ofURA3^(−723 to −224) 61-67-CPK005-G 61-67-CPK008-G URA3^(−723 to 701) andURA3^(−223 to 701) purified (SEQ ID NO: 27) (SEQ ID NO: 30) PCR products100 ng each of ERG13 and 61-67-CPK032-G 61-67-CPK052-G ERG13-P_(GAL)P_(GAL) purified PCR products (SEQ ID NO: 49) (SEQ ID NO: 57) 3 100 ngeach of ERG13-P_(GAL) 61-67-CPK031-G 61-67-CPK032-G ERG13-P_(GAL)- andtHMGR purified PCR (SEQ ID NO: 48) (SEQ ID NO: 49) tHMGR products

Plasmid pAM493 was generated by inserting the IDI1-P_(GAL)-tHMGR insertof vector pAM473 into vector pAM468. Vector pAM473 was generated byinserting DNA fragment IDI1-P_(GAL)-tHMGR, which comprises the ORF ofthe IDI1 gene of Saccharomyces cerevisiae (IDI1 nucleotide position 1 to1017) (IDI1), the genomic locus containing the divergent GAL1 and GAL10promoter of Saccharomyces cerevisiae (GAL1 nucleotide position −1 to−668) (P_(GAL)), and a truncated ORF of the HMG1 gene of Saccharomycescerevisiae (HMG1 nucleotide positions 1586 to 3323) (tHMGR), into theTOPO Zero Blunt II cloning vector. Vector pAM468 was generated byinserting DNA fragment ADE1^(−825 to 653), which comprises a segment ofthe wild-type ADE1 locus of Saccharomyces cerevisiae that extends fromnucleotide position −225 to position 653 and harbors a non-nativeinternal XmaI restriction site between bases −226 and −225, into theTOPO TA pCR2.1 cloning vector. DNA fragments IDI1-P_(GAL)-tHMGR andADE1^(−825 to 653) were generated by PCR amplification as outlined inTable 3. For the construction of pAM493, 400 ng of pAM473 and 100 ng ofpAM468 were digested to completion using XmaI restriction enzyme, DNAfragments corresponding to the IDI1-P_(GAL)-tHMGR insert and thelinearized pAM468 vector were gel purified, and 4 molar equivalents ofthe purified insert was ligated with 1 molar equivalent of the purifiedlinearized vector, yielding vector pAM493. FIG. 7C shows a map of theIDI1-P_(GAL)-tHMGR insert, and SEQ ID NO: 11 shows the nucleotidesequence of the DNA fragment and the flanking ADE1 sequences.

TABLE 3 PCR amplifications performed to generate pAM493 PCR RoundTemplate Primer 1 Primer 2 PCR Product 1 100 ng of Y007 genomic DNA61-67-CPK009-G 61-67-CPK010-G ADE1^(−825 to −226) (SEQ ID NO: 31) (SEQID NO: 32) 61-67-CPK011-G 61-67-CPK012-G ADE1^(−225 to 653) (SEQ ID NO:33) (SEQ ID NO: 34) 100 ng of Y002 genomic DNA 61-67-CPK047-G61-67-CPK064-G IDI1 (SEQ ID NO: 54) (SEQ ID NO: 69) 61-67-CPK052-G61-67-CPK065-G P_(GAL) (SEQ ID NO: 57) (SEQ ID NO: 70) 61-67-CPK031-G61-67-CPK053-G tHMGR (SEQ ID NO: 48) (SEQ ID NO: 58) 2 100 ng each ofADE1^(−825 to −226) 61-67-CPK009-G 61-67-CPK012-G ADE1^(−825 to 653) andADE1^(−225 to 653) purified PCR (SEQ ID NO: 31) (SEQ ID NO: 34) products100 ng each of IDI1 and P_(GAL) 61-67-CPK047-G 61-67-CPK052-GIDI1-P_(GAL) purified PCR products (SEQ ID NO: 54) (SEQ ID NO: 57) 3 100ng each of IDI1-P_(GAL) and 61-67-CPK031-G 61-67-CPK047-G IDI1-P_(GAL)-tHMGR purified PCR products (SEQ ID NO: 48) (SEQ ID NO: 54) tHMGR

Plasmid pAM495 was generated by inserting the ERG10-P_(GAL)-ERG12 insertof pAM474 into vector pAM469. Vector pAM474 was generated by insertingDNA fragment ERG10-P_(GAL)-ERG12, which comprises the ORF of the ERG10gene of Saccharomyces cerevisiae (ERG10 nucleotide position 1 to 1347)(ERG10), the genomic locus containing the divergent GAL1 and GAL10promoter of Saccharomyces cerevisiae (GAL1 nucleotide position −1 to−668) (P_(GAL)), and the ORF of the ERG12 gene of Saccharomycescerevisiae (ERG12 nucleotide position 1 to 1482) (ERG12), into the TOPOZero Blunt II cloning vector. Vector pAM469 was generated by insertingDNA fragment HIS3^(−32 to −1000)-HISMX-HIS3^(504 to −1103), whichcomprises two segments of the HIS locus of Saccharomyces cerevisiae thatextend from nucleotide position −32 to position −1000 and fromnucleotide position 504 to position 1103, a HISMX marker, and anon-native XmaI restriction site between the HIS3^(504 to −1103)sequence and the HISMX marker, into the TOPO TA pCR2.1 cloning vector.DNA fragments ERG10-P_(GAL)-ERG12 andHIS3^(−32 to −1000)-HISMX-HIS3^(504 to −1103) were generated by PCRamplification as outlined in Table 4. For construction of pAM495, 400 ngof pAM474 and 100 ng of pAM469 were digested to completion using XmaIrestriction enzyme, DNA fragments corresponding to theERG10-P_(GAL)-ERG12 insert and the linearized pAM469 vector were gelpurified, and 4 molar equivalents of the purified insert was ligatedwith 1 molar equivalent of the purified linearized vector, yieldingvector pAM495. FIG. 7D shows a map of the ERG10-P_(GAL)-ERG12 insert,and SEQ ID NO: 12 shows the nucleotide sequence of the DNA fragment andthe flanking HIS3 sequences.

TABLE 4 PCR reactions performed to generate pAM495 PCR Round TemplatePrimer 1 Primer 2 PCR Product 1 100 ng of Y007 genomic 61-67-CPK013-G61-67-CPK014alt-G HIS3^(−32 to −1000) DNA (SEQ ID NO: 35) (SEQ ID NO:36) 61-67-CPK017-G 61-67-CPK018-G HIS3^(504 to −1103) (SEQ ID NO: 39)(SEQ ID NO: 40) 61-67-CPK035-G 61-67-CPK056-G ERG10 (SEQ ID NO: 50) (SEQID NO: 61) 61-67-CPK057-G 61-67-CPK058-G P_(GAL) (SEQ ID NO: 62) (SEQ IDNO: 63) 61-67-CPK040-G 61-67-CPK059-G ERG12 (SEQ ID NO: 51) (SEQ ID NO:64) 10 ng of plasmid pAM330 61-67-CPK015alt-G 61-67-CPK016-G HISMX DNA** (SEQ ID NO: 37) (SEQ ID NO: 38) 2 100 ng each of HIS3^(504 to −1103)61-67-CPK015alt-G 61-67-CPK018-G HISMX- HIS3^(504 to −1103) and HISMXPCR (SEQ ID NO: 37) (SEQ ID NO: 40) 1103 purified products 100 ng eachof ERG10 and 61-67-CPK035-G 61-67-CPK058-G ERG10-P_(GAL) P_(GAL)purified PCR (SEQ ID NO: 50) (SEQ ID NO: 63) products 3 100 ng each ofHIS3^(−32 to) ⁻¹⁰⁰⁰ 61-67-CPK013-G 61-67-CPK018-G HIS3^(−32 to −1000)-and HISMX- (SEQ ID NO: 35) (SEQ ID NO: 40) HISMX-HIS3^(504 to −1103)HIS3^(504 to −1103) purified PCR products 100 ng each of ERG10-61-67-CPK035-G 61-67-CPK040-G ERG10-P_(GAL)-ERG12 P_(GAL) and ERG12purified (SEQ ID NO: 50) (SEQ ID NO: 51) PCR products ** The HISMXmarker in pAM330 originated from pFA6a-HISMX6-PGAL1 as described by vanDijken et al. ((2000) Enzyme Microb. Technol. 26(9-10): 706-714).

Plasmid pAM497 was generated by inserting the ERG8-P_(GAL)-ERG19 insertof pAM475 into vector pAM470. Vector pAM475 was generated by insertingDNA fragment ERG8-P_(GAL)-ERG19, which comprises the ORF of the ERG8gene of Saccharomyces cerevisiae (ERG8 nucleotide position 1 to 1512)(ERG8), the genomic locus containing the divergent GAL1 and GAL10promoter of Saccharomyces cerevisiae (GAL1 nucleotide position −1 to−668) (P_(GAL)), and the ORF of the ERG19 gene of Saccharomycescerevisiae (ERG19 nucleotide position 1 to 1341) (ERG19), into the TOPOZero Blunt II cloning vector. Vector pAM470 was generated by insertingDNA fragment LEU2^(−100 to 450)-HISMX-LEU2^(1096 to 1770), whichcomprises two segments of the LEU2 locus of Saccharomyces cerevisiaethat extend from nucleotide position −100 to position 450 and fromnucleotide position 1096 to position 1770, a HISMX marker, and anon-native XmaI restriction site between the LEU2^(1096 to 1770)sequence and the HISMX marker, into the TOPO TA pCR2.1 cloning vector.DNA fragments ERG8-P_(GAL)-ERG19 andLEU2^(−100 to 450)-HISMX-LEU2^(1096 to 1770) were generated by PCRamplification as outlined in Table 5. For the construction of pAM497,400 ng of pAM475 and 100 ng of pAM470 were digested to completion usingXmaI restriction enzyme, DNA fragments corresponding to theERGS-P_(GAL)-ERG19 insert and the linearized pAM470 vector werepurified, and 4 molar equivalents of the purified insert was ligatedwith 1 molar equivalent of the purified linearized vector, yieldingvector pAM497. FIG. 7E for a map of the ERG8-P_(GAL)-ERG19 insert, andSEQ ID NO: 13 shows the nucleotide sequence of the DNA fragment and theflanking LEU2 sequences.

TABLE 5 PCR reactions performed to generate pAM497 PCR Round TemplatePrimer 1 Primer 2 PCR Product 1 100 ng of Y007 genomic 61-67-CPK019-G61-67-CPK020-G LEU2^(−100 to 450) DNA (SEQ ID NO: 41) (SEQ ID NO: 42)61-67-CPK023-G 61-67-CPK024-G LEU2^(1096 to 1770) (SEQ ID NO: 45) (SEQID NO: 46) 10 ng of plasmid pAM330 61-67-CPK021-G 61-67-CPK022-G HISMXDNA ** (SEQ ID NO: 43) (SEQ ID NO: 44) 100 ng of Y002 genomic61-67-CPK041-G 61-67-CPK060-G ERG8 DNA (SEQ ID NO: 52) (SEQ ID NO: 65)61-67-CPK061-G 61-67-CPK062-G P_(GAL) (SEQ ID NO: 66) (SEQ ID NO: 67)61-67-CPK046-G 61-67-CPK063-G ERG19 (SEQ ID NO: 53) (SEQ ID NO: 68) 2100 ng each of LEU2^(1096 to 1770) 61-67-CPK021-G 61-67-CPK024-GHISMX-LEU2^(1096 to 1770) and HISMX purified PCR (SEQ ID NO: 43) (SEQ IDNO: 46) products 100 ng each of ERG8 and 61-67-CPK041-G 61-67-CPK062-GERG8-P_(GAL) P_(GAL) purified PCR products (SEQ ID NO: 52) (SEQ ID NO:67) 3 100 ng of LEU2^(−100 to 450) and 61-67-CPK019-G 61-67-CPK024-GLEU2^(−100 to 450)- HISMX-LEU2^(1096 to 1770) (SEQ ID NO: 41) (SEQ IDNO: 46) HISMX-LEU2^(1096 to 1770) purified PCR products 100 ng each ofERG8-P_(GAL) 61-67-CPK041-G 61-67-CPK046-G ERG8-P_(GAL) and ERG19purified PCR (SEQ ID NO: 52) (SEQ ID NO: 53) ERG19 products ** The HISMXmarker in pAM330 originated from pFA6a-HISMX6-PGAL1 as described by vanDijken et al. ((2000) Enzyme Microb. Technol. 26(9-10): 706-714).

Plasmid pAM584 was generated by inserting DNA fragmentGAL7^(4 to 1021)-HPH-GAL1^(1637 to 2587) into the TOPO ZERO Blunt IIcloning vector (Invitrogen, Carlsbad, Calif.). DNA fragmentGAL7^(4 to 1021)-HPH-GAL1^(1637 to 2587) comprises a segment of the ORFof the GAL7 gene of Saccharomyces cerevisiae (GAL7 nucleotide positions4 to 1021) (GAL7^(4 to 1021)), the hygromycin resistance cassette (HPH),and a segment of the 3′ untranslated region (UTR) of the GAL1 gene ofSaccharomyces cerevisiae (GAL1 nucleotide positions 1637 to 2587). TheDNA fragment was generated by PCR amplification as outlined in Table 6.FIG. 7F shows a map and SEQ ID NO: 102 the nucleotide sequence of DNAfragment GAL7^(4 to 1021)-HPH-GAL1^(1637 to 2587).

TABLE 6 PCR reactions performed to generate pAM584 PCR Round TemplatePrimer 1 Primer 2 PCR Product 1 100 ng of Y002 genomic 91-014-CPK236-G91-014-CPK237-G GAL7^(4 to 1021) DNA (SEQ ID NO: 126) (SEQ ID NO: 127)91-014-CPK232-G 91-014-CPK233-G GAL1^(1637 to 2587) (SEQ ID NO: 124)(SEQ ID NO: 125) 10 ng of plasmid pAM547 91-014-CPK231-G 91-014-CPK238-GHPH DNA ** (SEQ ID NO: 123) (SEQ ID NO: 128) 2 100 ng each ofGAL7^(4 to 1021) 91-014-CPK231-G 91-014-CPK236-G GAL7^(4 to 1021)- andHPH purified PCR (SEQ ID NO: 123) (SEQ ID NO: 126) HPH products 3 100 ngof each 91-014-CPK233-G 91-014-CPK236-G GAL7^(4 to 1021)-GAL1^(1637 to 2587) and (SEQ ID NO: 125) (SEQ ID NO: 126) HPH-GAL7^(4 to 1021)-HPH GAL1^(1637 to 2587) purified PCR products **Plasmid pAM547 was generated synthetically, and comprises the HPHcassette, which consists of the coding sequence for the hygromycin Bphosphotransferase of Escherichia coli flanked by the promoter andterminator of the Tef1 gene of Kluyveromyces lactis.

Integration cassette natA-P_(CTR3) ^(−1 to −734) was generated by PCRamplifying the natA marker using primers PW287-002-CPK1217 (SEQ ID NO:104) and DE_PW91-027-CPK262-G (SEQ ID NO: 99) and using plasmid DNAcomprising the TEF1 promoter and terminator of Kluyveromyces lactis(GenBank accession CR382122 REGIONS:788874 . . . 789380 and 787141 . . .787496, respectively) and the nat resistance marker. In addition, thepromoter of the CTR3 gene of Saccharomyces cerevisiae was PCR amplifiedfrom Y002 genomic DNA from positions −1 to −734 using primersPW287-002-CPK1232 (SEQ ID NO: 100) and DE_PW91-027-CPK263-G (SEQ ID NO:101). The 2 PCR products were stitched together in a secondary PCRreaction using 25 ng of each of the gel purified PCR fragments andprimers PW287-002-CPK1217 and PW287-002-CPK1232, yielding integrationcassette natA-P_(CTR3) ^(−1 to −734).

Additional recombinant integration cassettes were generated by stitchingRABits. RABits were generated by inserting DNA fragments of interest(MULEs) into the pMULE Entry vector.

The pMULE Entry vector was PCR amplified using primers K162 (SEQ ID NO:73) and K163 (SEQ ID NO: 74) and pRYSE Entry vector 8 (SEQ ID NO: 14) astemplate. The reaction mixture was resolved by gel electrophoresis, andthe approximately 2.2 kb vector backbone was gel purified. A DNAfragment comprising the lacZ coding sequence was generated by digestingto completion pRYSE Entry vector 8 using SchI restriction enzyme, heatinactivating the enzyme (20 min at 65° C.), resolving the reactionmixture by gel electrophoresis, and gel purifying the approximately 0.5kb DNA fragment. The purified DNA fragment comprising the lacZ codingsequence was ligated with the purified vector backbone, yielding thepMULE Entry vector (see FIG. 8 for a plasmid map).

MULEs were PCR amplified using templates and primers as outlined inTable 7. PCR amplifications were done using the Phusion DNA polymerase(New England Biolabs, Ipswich, Mass.) as per manufacturer's suggestedprotocol.

TABLE 7 Amplified MULEs MULE Primers Template Size (bp) 5′ ERG20-YD-198-75A (SEQ ID NO: 94) pAM489 1,012 pGAL1/10 YD-198-75B (SEQ ID NO:95) mIS2081 YD-198-75F (SEQ ID NO: 96) Genetrix2081 1,836 YD-198-75H(SEQ ID NO: 98) mIS2081T YD-198-75G (SEQ ID NO: 97) Genetrix2081 1,668YD-198-75H (SEQ ID NO: 98) tTDH3 RYSE 4 (SEQ ID NO: 77) RABit63 * 311RYSE 7 (SEQ ID NO: 78) Hyg YD-198-75L (SEQ ID NO: 105) RABit21 * 1,962YD-198-75M (SEQ ID NO: 106) TRP1 YD-198-75N (SEQ ID NO: 107) pAM489 586YD-198-75O (SEQ ID NO: 108) * RABit21 comprises SEQ ID NO: 15, andRABit63 comprises SEQ ID NO: 16.

RABits were generated by inserting the MULEs into the pMULE Entryvector. The PCR reactions were resolved by gel electrophoresis, theMULEs were gel purified, the purified MULEs were treated with T4polynucleotide kinase (PNK) (New England Biolabs, Ipswich, Mass.) as permanufacturer's suggested protocol, and the PNK was heat inactivated at65° C. for 20 minutes. The pMULE Entry vector was digested to completionusing SchI restriction enzyme, the approximately 2.2 kb pMULE Entryvector backbones (lacking lacZ) was gel purified, the purified pMULEEntry vector backbone was treated with Antarctic Phosphatase (NewEngland Biolabs, Ipswich, Mass.), and the phosphatase was heatinactivated at 65° C. for 20 minutes. The pMULE Entry vector backbonewas ligated with each of the amplified MULEs, yielding RABits.

RABits to be stitched (Table 8) were placed together in one tube (333fmole of each RABit) and digested to completion using LguI restrictionenzyme (Fermentas, Glen Burnie, Md.). The restriction enzyme was heatinactivated for 20 minutes at 65° C. The RABit digestion reactions weresplit into three 30 uL reactions; water, buffer, dNTPs, and DNApolymerase were added to each reaction mixture, and a first round of PCRamplification was initiated. Samples were placed on ice, 0.5 uM of eachterminal primer (Table 8) were added to the reaction mixtures, and asecond round of PCR amplification was performed. The three PCR reactionmixtures were combined in one tube, the reaction mixtures were resolvedby gel electrophoresis, and the PCR products were gel purified. FIG. 7Gshows a map of the integration cassettes.

TABLE 8 PCR Amplification of Integration Cassettes Integration TerminalPrimers for Cassette RABits to be combined 2^(nd) Round PCRamplification i00280 5′ ERG20-pGAL1/10-mIS2081-tTDH3-Hyg-TRP1 YD-198-75A(SEQ ID NO: 94) i00281 5′ ERG20-pGAL1/10-mIS2081T-tTDH3-Hyg-TRP1YD-198-75O (SEQ ID NO: 108) The first round of PCR amplification wasperformed as follows: one cycle of denature at 98° C. for 2 minutes; 5cycles of denature at 98° C. for 30 seconds and anneal/extend at 72° C.for 30 seconds per kilobase PCR product. The second round of PCRamplification was performed as follows: one cycle of denature at 98° C.for 2 minutes; 35 rounds of denature at 98° C. for 12 seconds andanneal/extend at 72° C. for 20 seconds per kilobase PCR product; onecycle of final extend at 72° C. for 7 minutes; and a final hold at 4° C.

Example 3

This example describes methods for generating Saccharomyces cerevisiaestrains expressing heterologous isoprene synthases.

Saccharomyces cerevisiae strains CEN.PK2-1C (Y002) (MATA; ura3-52;trp1-289; leu2-3,112; his3A1; MAL2-8C; SUC2) and CEN.PK2-1D (Y003)(MATalpha; ura3-52; trp1-289; leu2-3,112; his3A1; MAL2-8C; SUC2) (vanDijken et al. (2000) Enzyme Microb. Technol. 26(9-10):706-714) wereprepared for introduction of inducible MEV pathway genes by replacingthe ERG9 promoter with the Saccharomyces cerevisiae MET3 promoter, andthe ADE1 ORF with the Candida glabrata LEU2 gene (CgLEU2). This was doneby PCR amplifying the KanMX-PMET3 region of vector pAM328 (SEQ ID NO:17) using primers 50-56-pw100-G (SEQ ID NO: 19) and 50-56-pw101-G (SEQID NO: 20), which include 45 base pairs of homology to the native ERG9promoter, transforming 10 μg of the resulting PCR product intoexponentially growing Y002 and Y003 cells using 40% w/w PolyetheleneGlycol 3350 (Sigma-Aldrich, St. Louis, Mo.), 100 mM Lithium Acetate(Sigma-Aldrich, St. Louis, Mo.), and 10 μg Salmon Sperm DNA (InvitrogenCorp., Carlsbad, Calif.), and incubating the cells at 30° C. for 30minutes followed by heat shocking them at 42° C. for 30 minutes(Schiestl and Gietz. (1989) Curr. Genet. 16, 339-346). Positiverecombinants were identified by their ability to grow on rich mediumcontaining 0.5 μg/ml Geneticin (Invitrogen Corp., Carlsbad, Calif.), andselected colonies were confirmed by diagnostic PCR. The resultant cloneswere given the designation Y93 (MAT A) and Y94 (MAT alpha). The 3.5 kbCgLEU2 genomic locus was then amplified from Candida glabrata genomicDNA (ATCC, Manassas, Va.) using primers 61-67-CPK066-G (SEQ ID NO: 71)and 61-67-CPK067-G (SEQ ID NO: 72), which contain 50 base pairs offlanking homology to the ADE1 ORF, and 10 μg of the resulting PCRproduct were transformed into exponentially growing Y93 and Y94 cells,positive recombinants were selected for growth in the absence of leucinesupplementation, and selected clones were confirmed by diagnostic PCR.The resultant clones were given the designation Y176 (MAT A) and Y177(MAT alpha).

Strain Y188 was generated by digesting 2 μg of pAM491 and pAM495 plasmidDNA to completion using PmeI restriction endonucleose (New EnglandBiolabs, Beverly, Mass.), and introducing the purified DNA inserts intoexponentially growing Y176 cells. Positive recombinants were selectedfor by growth on medium lacking uracil and histidine, and integrationinto the correct genomic locus was confirmed by diagnostic PCR.

Strain Y189 was generated by digesting 2 μg of pAM489 and pAM497 plasmidDNA to completion using PmeI restriction endonuclease, and introducingthe purified DNA inserts into exponentially growing Y177 cells. Positiverecombinants were selected for by growth on medium lacking tryptophanand histidine, and integration into the correct genomic locus wasconfirmed by diagnostic PCR.

Strain Y238 was generated by mixing approximately 1×10⁷ cells fromstrains Y188 and Y189 on a YPD medium plate for 6 hours at roomtemperature to allow for mating, plating the mixed cell culture tomedium lacking histidine, uracil, and tryptophan to select for growth ofdiploid cells, digesting 2 μg of pAM493 plasmid DNA to completion usingPmeI restriction endonuclease, and introducing the purified DNA insertinto the exponentially growing diploid cells. Positive recombinants wereselected for by growth on medium lacking adenine, and integration intothe correct genomic locus was confirmed by diagnostic PCR.

Strains Y210 (Mat A) and Y211 (MAT alpha) were generated by sporulatingstrain Y238 in 2% Potassium Acetate and 0.02% Raffinose liquid medium,isolating approximately 200 genetic tetrads using a Singer InstrumentsMSM300 series micromanipulator (Singer Instrument LTD, Somerset, UK),identifying independent genetic isolates containing the appropriatecomplement of introduced genetic material by their ability to grow inthe absence of adenine, histidine, uracil, and tryptophan, andconfirming the integration of all introduced DNA by diagnostic PCR.

Strain Y258 was generated by transforming strain Y211 with pAM404plasmid DNA. Host cell transformants were selected on synthetic definedmedia, containing 2% glucose and all amino acids except leucine(SM-glu). Single colonies were transferred to culture vials containing 5mL of liquid SM-glu lacking leucine, and the cultures were incubated byshaking at 30° C. until growth reached stationary phase. The cells werestored at −80° C. in cryo-vials in 1 mL frozen aliquots made up of 400uL 50% sterile glycerol and 600 uL liquid culture.

Strains Y225 (MAT A) and Y227 (MAT alpha) were generated by transformingexponentially growing Y210 and Y211 cells, respectively, with 2 μg ofpAM426 (SEQ ID NO: 18), which comprises a GAL1 promoter operably linkedto the coding sequence of an amorpha-4,11-diene synthase gene that iscodon-optimized for expression in Saccharomyces cerevisiae (Merke et al.(2000) Ach. Biochem. Biophys. 381:173-180). Host cell transformants wereselected on complete synthetic defined media lacking leucine.

Strain Y337 was generated from strain Y227 by rendering the strainunable to catabolize galactose. To this end, pAM584 plasmid DNA wasdigested to completion using PmeI restriction endonuclease, and thepurified DNA insert GAL7^(4 to 1021)-HPH-GAL1^(637 to 2587) wasintroduced into exponentially growing Y227 cells. Positive recombinantswere selected for by growth on solid medium lacking adenine, leucine,lysine, histidine, methionine, uracil, and tryptophan, and containing900 μg/mL hygromycin B (Sigma, St. Louis, Mo.). Integration into thecorrect genomic locus was confirmed by diagnostic PCR and by testing thestrain for inability to use galactose as a carbon source.

Strain Y615 was generated from strain Y337 by replacing the URA3 openreading frame with the hisG open reading frame from Salmonella sp. A DNAfragment comprising the hisG open reading frame was PCR amplified frompAM840 using primers 100-150-KB034-G (SEQ ID NO: 109) and100-150-KB039-G (SEQ ID NO: 110), and the purified DNA fragment wasintroduced into exponentially growing Y337 cells. Positive transformantswere selected for their ability to grow on medium containing5-fluoroorotic acid and for their inability to grow on medium lackinguracil. Integration into the correct genomic locus was confirmed bydiagnostic PCR.

Strain Y1775 was generated from strain Y615 by replacing the kanMXmarker with the URA3 marker. A DNA fragment encoding the S. cerevisiaeURA3 auxotrophic marker was PCR amplified from pAM64 (SEQ ID NO: 103)using primers PW-191-046-CPK1212-G (SEQ ID NO: 111) andPW-191-046-CPK1213-G (SEQ ID NO: 112), and the purified DNA fragment wasintroduced into exponentially growing Y615 cells. Positive recombinantswere selected for by growth on medium lacking adenine, leucine, lysine,histidine, methionine, uracil, and tryptophan. Integration into thecorrect genomic locus was confirmed by diagnostic PCR.

Strain Y1791 was generated from strain Y1775 by restoring the ERG9locus. A DNA fragment comprising the ERG9 open reading frame was PCRamplified from Y002 genomic DNA using primers PW-191-015-CPK947-G (SEQID NO: 113) and PW-191-015-CPK950-G (SEQ ID NO: 114), and the purifiedDNA fragment was introduced into exponentially growing Y1775 cells.Positive transformants we selected for their ability to grow on mediumcontaining 5-fluoroorotic acid and for their inability to grow on mediumlacking uracil. Integration into the correct genomic locus was confirmedby diagnostic PCR.

Strain Y1856 was generated from strain Y1791 by restoring the GAL1,GAL10, and GAL7 locus. A DNA fragment comprising the GAL1, GAL10, andGAL7 genomic region was PCR amplified from Y002 genomic DNA usingprimers PW-91-093-CPK453-G (SEQ ID NO: 115) and PW-091-144-CPK689-G (SEQID NO: 116), and the purified DNA fragment was introduced intoexponentially growing Y1791 cells. Positive recombinants were selectedfor by growth on medium containing 20 g/L glacatose and their inabilityto grow on medium containing 900 μg/mL hygromycin B.

Strain Y1857 was generated from strain Y1856 by disrupting theP_(GAL10)-ERG20 locus. A DNA fragment encoding the S. cerevisiae URA3auxotrophic marker was PCR amplified from pAM64 (SEQ ID NO: 130) usingprimers PW-287-002-CPK1215-G (SEQ ID NO: 117) and PW-287-002-CPK1216-G(SEQ ID NO: 118), and the purified DNA fragment was introduced intoexponentially growing Y1856 cells. Positive recombinants were selectedfor by growth on medium lacking adenine, leucine, lysine, histidine,methionine, uracil, and tryptophan. Integration into the correct genomiclocus was confirmed by diagnostic PCR.

Strain Y1895 was generated from strain Y1857 by curing the strain frompAM426. Strain Y1857 was propagated in rich Yeast Extract PeptoneDextrose (YPD) medium contain 0.5% leucine (w/v) for 5 days. Every 12hours, fresh YPD 0.5% LEU was inoculated to an 0D600 of 0.05 using theprevious 12 hour growth. After 5 days, the cells were plated on YPD 0.5%leucine agar medium and incubated at 30° C. for 2 days. Cured cells wereidentified by their ability to grow on minimal medium containing leucineand their inability to grow on medium lacking leucine.

Strain Y736 was generated from strain Y227 by replacing the URA3 markerwith a hisG marker. To this end, the hisG marker was PCR amplified usingprimers KB34 (SEQ ID NO: 75) and KB39 (SEQ ID NO: 76) and plasmid pAM840as template. Exponentially growing Y211 cells were transformed with thePCR mixture and were then plated on YPD overnight. Host celltransformants were selected by replica plating cells from the YPD platesonto Complete Synthetic Medium (CSM) solid media lacking methionine andleucine and containing 0.1% 5-FOA and 0.1 mg/ml uracil.

Strain Y737 was generated from strain Y736 by transforming exponentiallygrowing cells with pAM940, and selecting host cell transformants on CSMsolid media lacking leucine and uracil.

Strain Y846 was generated from strain Y737 by curing the strain ofpAM426. To this end, strain Y737 was grown for 3 successive nights inrich media supplemented with 6× the usual concentration of leucine,being diluted each morning 100×. The cells were then plated out towell-separated single colonies on rich media and replica plated ontominimal media lacking leucine. Colonies that grew on rich media but didnot grow in the absence of leucine were picked, grown, and tested by PCRto verify that the plasmid was no longer present. One such positivecolonie was stocked as Y846.

Strain Y1858 was generated from strain Y1895 by heterologous expressionof an isoprene synthase. To this end, exponentially growing Y1895 cellswere transformed with expression plasmid pAM1547. Host celltransformants were selected on CSM agar lacking methionine and leucineand containing 2% glucose.

Strain Y1859 was generated from strain Y1895 by heterologous expressionof an isoprene synthase. To this end, exponentially growing Y1895 cellswere transformed with expression plasmid pAM1548. Host celltransformants were selected on CSM agar lacking methionine and leucineand containing 2% glucose.

Strain Y1860 was generated from strain Y1895 by heterologous expressionof an isoprene synthase. To this end, exponentially growing Y1895 cellswere transformed with expression plasmid pAM1549. Host celltransformants were selected on CSM agar lacking methionine and leucineand containing 2% glucose.

Strain Y1861 was generated from strain Y1895 by heterologous expressionof an isoprene synthase. To this end, exponentially growing Y1895 cellswere transformed with expression plasmid pAM1550. Host celltransformants were selected on CSM agar lacking methionine and leucineand containing 2% glucose.

Strain Y1713 was generated from strain Y846 by replacing the ERG20 genewith the coding sequence for an isoprene synthase. To this end,exponentially growing Y846 cells were transformed with integrationcassette i00280. Host cell transformants were selected on YPD agarcontaining 2% glucose and 300 μg/mL hygromycin B (A.G. Scientific, SanDiego, Calif.).

Strain Y1714 was generated from strain Y846 by replacing the ERG20 genewith a coding sequence for a truncated isoprene synthase. To this end,exponentially growing Y846 cells were transformed with integrationcassette i00281. Host cell transformants were selected on YPD agarcontaining 2% glucose and 300 μg/mL hygromycin B (A.G. Scientific, SanDiego, Calif.).

Strain Y1732 was generated from strain Y846 by heterologous expressionof an isoprene synthase. To this end, exponentially growing Y846 cellswere transformed with expression plasmid pAM1549. Host celltransformants were selected on CSM agar lacking methionine and leucineand containing 2% glucose.

Strain Y1733 was generated from strain Y846 by heterologous expressionof a truncated isoprene synthase. To this end, exponentially growingY846 cells were transformed with expression plasmid pAM1550. Host celltransformants were selected on CSM agar lacking methionine and leucineand containing 2% glucose.

Strain Y1837 was generated by transforming exponentially growing Y1713cells with expression plasmid pAM1549. Host cell transformants wereselected on CSM agar lacking methionine and leucine and containing 2%glucose.

Strain Y1838 was generated by transforming exponentially growing Y1714cells with expression plasmid pAM1550. Host cell transformants wereselected on CSM agar lacking methionine and leucine and containing 2%glucose.

Strain 1907 was generated from strain Y1860 by replacing the ERG20promoter with the nourseothricin resistance marker (natA) and the copperrepressible promoter of the CTR3 gene of Saccharomyces cerevisiae. Tothis end, exponentially growing Y1860 cells were transformed with 200 μgof the integration cassette natA-P_(CTR3) ^(−1 to −734). Host celltransformants were selected for by growth on rich YPD medium containing300 μg/mL nourseothricin (Werner BioAgents, Jena, Germany).

Example 4

This example describes methods for producing isoprene in Saccharomycescerevisiae host strains.

Single colonies of host cell transformants were transferred to culturevials containing 5 mL of Bird Seed Medium containing 0.25 uM CuSO₄. Thefollowing day, 20 mL of Bird Production Medium containing 1.8%galactose, 0.2% glucose, and 32 uM CuSO₄, with 4 mL isopropylmyristate,was inoculated with host cell transformant Y1858, Y1859, Y1860, or Y1861to an OD₆₀₀ of 0.05. Similarly, 20 mL of Bird Production Mediumcontaining 1.8% galactose and 0.2% glucose, with 4 mLisopropylmyristate, was inoculated with isolates #3, 6, or 9 of hostcell transformant Y1907 to an OD₆₀₀ of 0.05. To the Y1907 culture, 0.25uM CuSO₄, 50 uM CuSO₄, or 150 uM CuSO₄ was added. The shake flasks weresealed for anaerobic growth and incubated at 30° C. on a rotary shakerat 200 rpm.

After 72 hours of growth, the cultures were assayed for cell growth. Atthe same time, 200 uL of isopropylmyristate was removed from each flaskand were injected directly on an Agilent 7980 gas chromatograph equippedwith a flame ionization detector. To expedite run times, the temperatureprogram and column matrix were modified to achieve optimal resolutionand the shortest overall runtime (15.0 min). Each 2 μL sample was split10:1 and was separated using a Varian fused silica CP-PoraBond U PLOT(25 m×0.32 mm×7 um; length×width×film thickness) column with hydrogen asthe carrier gas. The temperature program for the analysis was asfollows: the column was initially held at 100° C. for 1 minute, followedby a temperature gradient of 10° C./min to a temperature of 140° C.,followed by a temperature gradient of 40° C./min to a temperature of250° C., followed by holding the column at 250° C. for 6.5 min Underthese conditions, isoprene elutes at 5.8 minutes.

What is claimed is:
 1. A gaseous isoprene composition comprisingisoprene, carbon dioxide and water, wherein the isoprene is present inan amount between about 0.1% and about 15% by volume; wherein the carbondioxide is in an amount greater than about 0.04% by volume; wherein thewater is in an amount greater than about 70% of its saturation amount;and wherein the gaseous isoprene composition comprises 1 part permillion or less of C₂-C₅ alkynes.
 2. The gaseous isoprene composition ofclaim 1, wherein the gaseous composition comprises less than about 3% byweight of water.
 3. The gaseous isoprene composition of claim 1, whereinthe gaseous composition further comprises oxygen in an amount betweenabout 1% and about 20% by volume.
 4. The gaseous isoprene composition ofclaim 1, wherein the gaseous composition further comprises nitrogen inan amount greater than about 50% by volume.
 5. The gaseous isoprenecomposition of claim 1, wherein the gaseous composition furthercomprises argon in an amount less than about 0.9% by volume.
 6. Thegaseous isoprene composition of claim 1, wherein the gaseous compositionfurther comprises argon in an amount greater than about 1.0% by volume.7. The gaseous isoprene composition of claim 1, wherein the gaseouscomposition further comprises ethanol in an amount less than about 0.5%by volume.
 8. The gaseous isoprene composition of claim 1, wherein thegaseous composition further comprises ethanol in an amount more thanabout 1% by volume.
 9. The gaseous isoprene composition of claim 1,wherein the gaseous composition further comprises 1 part per million orless of cyclopentadiene.
 10. The gaseous isoprene composition of claim1, wherein the gaseous composition further comprises 1 part per millionor less of piperylene.
 11. The gaseous isoprene composition of claim 1,wherein the gaseous composition further comprises 1 part per million orless of 1,4-pentadiene.
 12. The gaseous isoprene composition of claim 1,wherein the isoprene is present in an amount between about 1% and about5% by volume.
 13. The gaseous isoprene composition of claim 1, whereinthe isoprene is present in an amount between about 1% and about 10% byvolume.
 14. The gaseous isoprene composition of claim 1, wherein theisoprene is present in an amount between about 5% and about 10% byvolume.
 15. The gaseous isoprene composition of claim 1, wherein thecarbon dioxide is present in an amount that is greater than about 5% byvolume.
 16. The gaseous isoprene composition of claim 1, wherein thecarbon dioxide is present in an amount that is greater than about 10% byvolume.
 17. The gaseous isoprene composition of claim 1, wherein thecarbon dioxide is present in an amount that is between about 1% andabout 35% by volume.
 18. The gaseous isoprene composition of claim 1,wherein the gaseous composition further comprises oxygen in an amountless than about 5% by volume.
 19. The gaseous isoprene composition ofclaim 1, wherein the gaseous composition further comprises oxygen in anamount less than about 2% by volume.
 20. The gaseous isoprenecomposition of claim 1, wherein the gaseous isoprene composition isobtained by using a plurality of genetically modified host cells capableof making isoprene via the DXP and/or MEV pathway.